Process of making a compound by forming a polymer from a template drug

ABSTRACT

A method of forming polymers in the presence of nucleic acid using template polymerization. Also, a method of having the polymerization occur in heterophase systems. These methods can be used for the delivery of nucleic acids, for condensing the nucleic acid, for forming nucleic acid binding polymers, for forming supramolecular complexes containing nucleic acid and polymer, and for forming an interpolyelectrolyte complex.

BACKGROUND

[0001] Polymers are used for drug delivery for a variety of therapeuticpurposes. Polymers have also been used for the delivery of nucleic acids(polynucleotides and oligonucleotides) to cells for therapeutic purposesthat have been termed gene therapy or anti-sense therapy. One of theseveral methods of nucleic acid delivery to the cells is the use ofDNA-polycations complexes. It was shown that cationic proteins likehistones and protamines or synthetic polymers like polylysine,polyarginine, polyomithine, DEAE dextran, polybrene, andpolyethylenimine were effective intracellular delivery agents whilesmall polycations like spermine were ineffective. (Felgner, P. L. (1990)Advanced Drug Delivery Rev. 5, 163-187; Boussif, O., Lezoualch, F.,Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., & Behr, J. P.(1995) Proc. Natl. Acad. Sci. USA 92, 7297-7301) The mechanism by whichpolycations facilitate uptake of DNA is not completely understood butthe following are some important principles:

[0002] 1) Polycations Provide Attachment of DNA to the Target CellSurface:

[0003] The polymer forms a cross-bridge between the polyanionic nucleicacids and the polyanionic surfaces of the cells. As a result the mainmechanism of DNA translocation to the intracellular space might benon-specific adsorptive endocytosis which may be more effective thenliquid endocytosis or receptor-mediated endocytosis. Furthermore,polycations are a very convenient linker for attaching specificreceptors to DNA and as result, DNA-polycation complexes can be targetedto specific cell types.(Perales, J. C., Ferkol, T. Molas, M. & Hanson,W. (1994) Eur. J. Biochem. 226, 255-266; Cotten, M., Wagner, E. &Birustiel, M. L. (1993) Methods in Enzymology 217, 618-644; Wagner, E.,Curiel, D., & Cotten, M. (1994) Advanced Drug Delivery Rev. 1 14,113-135). 2) Polycations protect DNA in complexes against nucleasedegradation (Chiou, H. C., Tangco, M. V. Levine, S. M., Robertson, D.,Kormis, K., Wu, C. H., & Wu, G. Y. (1994) Nucleic Acids Res. 22,5439-5446). This is important for both extra- and intracellularpreservation of DNA. The endocytic step in the intracellular uptake ofDNA-polycation complexes is suggested by results in which DNA expressionis only obtained by incorporating a mild hypertonic lysis step (eitherglycerol or DMSO) (Lopata, M. A., D. Clevland, W., & Sollner-Webb, B.(1984) Nucleic Acids Res. 12, 5707-5717; Golub, E.I., Kim, H. & Volsky,D. J. (1989) Nucleic Acid Res. 17, 4902). Gene expression is alsoenabled or increased by preventing endosome acidification with NH₄CI orchloroquine (Luthman, H. & Magnusson, G. (1983) Nucleic Acids Res. 11,1295-1300). Polyethylenimine which facilitates gene expression withoutadditional treatments probably disrupts endosomal function itself(Boussif, O., Lezoualch, F., Zanta, M. A., Mergny, M.D., Scherman, D.,Demeneix, B., & Behr, J.P. (1995) Proc. Natl. Acad. Sci. USA 92,7297-7301). Disruption of endosomal function has also been accomplishedby linking to the polycation endosomal-disruptive agents such as fusionpeptides or adenoviruses (Zauner, W., Blaas, D., Kuechler, E., Wagner,E., (1995) J. Virology 69, 1085-1092; Wagner, E., Plank, C., Zatloukal,K., Cotten, M., & Birmstiel, M. L. (1992) Proc. Natl. Acad. Sci. 89,7934-7938) (Fisher, K. J., & Wilson, J. M. (1994) Biochemical J. 299,49-58).

[0004] 3) Polycations Generate DNA Condensation:

[0005] The volume which one DNA molecule occupies in complex withpolycations is drastically lower than the volume of a free DNA molecule.The size of DNA/polymer complex is critical for gene delivery in vivo.In terms of intravenous injection, DNA needs to cross the endothelialbarrier and reach the parenchymal cells of interest. The largestendothelia fenestrae (holes in the endothelial barrier) occurs in theliver and have an average diameter 100 mn. The fenestrae size in otherorgans is much lower. The size of the DNA complexes is also importantfor the cellular uptake process. After binding to the target cells theDNA-polycation complex should be taken up by endocytosis. Since theendocytic vesicles have a homogenous internal diameter of about 100 nmin hepatocytes of similar size in other cell types, the DNA complexesneed to be smaller than 100 nm (Geuzze, H. J., Slot, J. W., Strous, G.J., Lodish, H. F., & Schwartz, A. L. (1982) J. Cell Biol. 92, 865-870).

[0006] Condensation of DNA

[0007] A significant number of multivalent cations with widely differentmolecular structures have been shown to induce the condensation of DNA.These include spermidine, spermine, Co(NH₃)63+, protamine, histone Hi,and polylysine. (Gosule, L. C. & Schellman, J. A. (1976) Nature 259,333-335; Chattoraj, D. K., Gosule, L. C. & Schellman, J. A. (1978) J.Mol. Biol. 121, 327-337; Had, N. V., Downing, K. H. & Balhorn, R. (1993)Biochem. Biophys. Res. Commun. 193, 1347-1354; Hsiang, M. W & Cole, R.D. (1977) Proc. Natl. Acad. Sci. USA 74, 4852-4856; Haynes, M., Garret,R. A. & Gratzer, W. B. (1970) Biochemistry 9, 4410-4416; Widom, J. &Baldwin, R. L. (1980) J. Mol. Biol. 144, 431-453.). Quantitativeanalysis has shown DNA condensation to be favored when 90% or more ofthe charges along the sugar-phosphate backbone are neutralized (Wilson,R. W. & Bloomfield, V. A. (1979) Biochemistry 18, 2192-2196). Dependingupon the concentration of the DNA condensation leads to three main typesof structures:

[0008] 1) In extremely dilute solution (about 1 ug/ml or below), longDNA molecules can undergo a monomolecular collapse and form structuresdescribed as toroid.

[0009] 2) In very dilute solution (about 10 ug/ml) microaggregates formwith short or long molecules and remain in suspension. Toroids, rods andsmall aggregates can be seen in such solution.

[0010] 3) In dilute solution (about 1 mg/ml, large aggregates are formedthat sediment readily. (Sicorav, J. -L., Pelta, J., & Livolant, F (1994)Biophysical Journal 67, 1387-1392).

[0011] Toroids have been considered an attractive form for gene deliverybecause they have the lowest size. While the size of DNA toroidsproduced within single preparations has been shown to vary considerably,toroid size is unaffected by the length of DNA being condensed. DNAmolecules from 400 bp to genomic length produce toroids similar in size(Bloomfield, V. A. (1991) Biopolymers 31, 1471-1481). Therefore onetoroid can include from one to several DNA molecules. The kinetics ofDNA collapse by polycations which resulted in toroids is very slow. Forexample DNA condensation by Co(NH3)6CI3 needs 2 hours at roomtemperature.(Arscott, P. G., Ma, C., & Bloomfield, V. A. (1995)Biopolymers 36, 345-364).

[0012] The mechanism of DNA condensation is not obvious. Theelectrostatic forces between unperturbed helices arise primarily from acounterion fluctuation mechanism requiring multivalent cations and playsthe major role in DNA condensation.(Riemer, S. C. & Bloomfield, V. A.(1978) Biopolymers 17, 789-794; Marquet, R. & Houssier, C. (1991) J.Biomol. Struct. Dynam. 9, 159-167; Nilsson, L. G., Guldbrand, L. &Nordenskjold L. (1991) Mol. Phys. 72, 177-192). The hydration forcespredominate over electrostatic forces when the DNA helices approachcloser then a few water diameters (Leikin, S., Parsegian, V. A., Rau,D.C. & Rand, R. P. (1993) Ann. Rev. Phys. Chem. 44, 369-395). In case ofDNA-polymeric polycation interactions, DNA condensation is a morecomplicated process than the case of low molecular weight polycations.Different polycationic proteins can generate toroid and rod formationwith different size DNA at a ratio of positive to negative charge of 0.4(Garciaramirez, M., & Subirana, J. A. (1994) Biopolymers 34, 285-292).It was shown by fluorescence microscopy that T4 DNA complexed withpolyarginine or histone can forms two types of structures; an elongatedstructure with a long axis length of about 350 mn (like free DNA) anddense spherical particles.(Minagawa, K., Matsuzawa, Y., Yshikawa, K.,Matsumoto, M., & Doi, M. (1991) FEBS Lett. 295, 60-67). Both forms existsimultaneously in the same solution. The reason for the co-existence ofthe two forms can be explained as an uneven distribution of thepolycation chains among the DNA molecules. The uneven distributiongenerates two thermodynamically favorable conformations. (Kabanov, A.V., & Kabanov, V. A. (1995) Bioconjugate Chem. 6, 7-20).

[0013] It was also shown that the electrophoretic mobility of DNA-polycation complexes can change from negative to positive in excess ofpolycation. It is likely that large polycations don't completely alignalong DNA but form polymer loops which interact with other DNAmolecules. The rapid aggregation and strong intermolecular forcesbetween different DNA molecules may prevent the slow adjustment betweenhelices needed to form tightly packed, orderly particles. Thisspecification describes a new approach, that we have termedPolynucleotide Template Polymerization, for overcoming this problem ofnonspecific aggregation and large DNA-polycation complex formation thatoccurs when polycation/DNA complexes are formed in DNA concentrationsthat are of practical value for polynucleotide transfer into cells andfor gene or antisense therapy.

SUMMARY OF INVENTION

[0014] A process for drug delivery is described in which polymerizationand chemical reaction processes are induced in the presence of the drugin order to deliver the drug or biologically active compound. Drugdelivery encompasses the delivery of a biologically active compound to acell. By “delivering” we mean that the drug becomes associated with thecell. The drug can be on the membrane of the cell or inside thecytoplasm, nucleus, or other organelle of the cell. The process ofdelivering a polynucleotide to a cell has also been commonly termed“transfection” or the process of “transfecting” and also it has beentermed “transformation”. A biologically active compound is a compoundhaving the potential to react with biological components.Pharmaceuticals, proteins, peptides and nucleic acids are examples ofbiologically active compounds. The template polymer can be a polyanionsuch as a nucleic acid. The polynucleotide could be used to produce achange in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic purposes is commonlycalled “gene therapy”.

[0015] A new method is described for forming condensed nucleic acid byhaving a chemical reaction take place in the presence of the nucleicacid. A process is also described of forming in the presence of thenucleic acid a polymer that has affinity to nucleic acid. Moreover, aprocess is described of forming an interpolyelectrolyte complexcontaining nucleic acids by having a chemical reaction take place in thepresence of the nucleic acid. In addition, the nucleic acid-bindingpolymer can form as a result of template polymerization. This obviouslyexcludes the formation of polymers such as proteins or nucleic acids orother derivatives that bind nucleic acid by Watson-Crick binding.

[0016] Previously, the occurrence of chemical reactions or the processof polymerization in the presence of the nucleic acid has beenassiduously avoided when delivering nucleic acid. Perhaps, this aroseout of concerns that the processes of chemical reactions orpolymerization would chemically modify the nucleic acid and therebyrender it not biologically active. Surprisingly, we show that we canperform polymerizations in the presence of nucleic acids withoutchemically modifying the nucleic acid and that the nucleic acid is stillfunctional. For example, a plasmid construct containing a promoter andthe reporter gene luciferase can still express as much luciferase asnative plasmid after transfection into cells.

[0017] The process of forming a polymer in the presence of nucleic acidhas several advantages. As FIG. 1 illustrates, aggregation andprecipitation of the nucleic acid can be avoided by having thepolymerization take place in the presence of the nucleic acid. Thisnewly described process enabled us to form supramolecular complexes ofnucleic acid and polymer rapidly, consistently, and at very highconcentrations of polynucleic acid. In fact, high concentration of thetemplate nucleic acid favors this process. In contrast, the previouslydescribed process of mixing a nucleic acid and an already-formedpolycation (such as polylysine) has to be done at very diluteconcentrations. In addition, the previously-described procedure requiresthat the mixing, salt and ionicity conditions must be carefullycontrolled as well. This explains why the use of polylysine-DNAcomplexes are not widely used for the transfer of DNA into cells and isonly done in a few laboratories.

[0018] The other advantage that flows from the newly described processof having polymerization take place in the presence of nucleic acid isthat polymers could form that would not be able to become associatedwith nucleic acids if the polymer was formed first. For example, thepolymerization process could result in a hydrophobic polymer that is notsoluble in aqueous solutions unless it is associated with nucleic acid.A hydrophobic moiety comprises a C6-C24 alkane, C6-C24 alkene, sterol,steroid, lipid, or hydrophobic hormone. Furthermore, the process ofhaving the polymerization taking place in organic solvents andheterophase systems enables more types and more defined types ofvesicles to be formed.

[0019] This process will enable supramolecular complexes to be moreeasily assembled. It will also enable novel and more defined complexesto be made. Yet another advantage that flows from this invention is thatnucleic acid/polymer complexes will be smaller. The size of DNA/polymercomplex is critical for gene delivery especially in vivo.

[0020] These processes can be used for transferring nucleic acids intocells or an organism such as for drug delivery. They may also be usedfor analytical methods or the construction of new materials. They mayalso be used for preparative methods such as in the purification ofnucleic acids. They are also useful for many types of recombinant DNAtechnology. For example, they may be used to generate sequence bindingmolecules and protect specific sequences from nuclease digestion.Protection of specific regions of DNA is useful in many applications forrecombinant DNA technology.

[0021] A preferred embodiment provides a method of making a compound fordelivery to a cell, comprising: forming a polymer in the presence of abiologically active drug.

[0022] Another preferred embodiment provides a method of making acompound for delivery to a cell, comprising: cross-linking a polymer inthe presence of a polyion, thereby forming a complex of polymer andpolyion; and, delivering the complex to the cell.

[0023] Another preferred embodiment provides a method of making acompound for delivery to a cell, comprising: modifying a molecule in thepresence of the polyion thereby providing a deliverable polyion.

[0024] Yet, another preferred embodiment provides a method of making acompound for delivery to a cell, comprising: mixing a polyion with afirst polymer and a second polymer thereby forming a deliverablecomplex.

[0025] Further objects, features, and advantages of the invention willbe apparent from the following detailed description when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is a comparison of pDNA following template polymerizationand complexation contrasted with preformed polycations binding andprecipitating.

[0027]FIG. 2 shows complexes ranging in size from 40-70 nanometers indiameter after being dried onto carbon grids and stained withmethylamine tungstate.

[0028]FIG. 3 illustrates template dependent polymerization of NLSPeptides using SDS-PAGE.

[0029]FIG. 4 illustrates the relationship between turbidity (anindication of aggregation) and the molar charge ratio of polylysine(PLL):plasmid DNA without and with 100 mM NaCl and without and withouttemplate polymerization/caging (indicated by +DTBP).

[0030]FIG. 5 illustrates the ability of dextran sulfate (DS) to enableethidium bromide to interact with plasmid DNA that has been complexedwith varying ratios of PLL/DNA (molar ratio of lysine residue to DNAbase) (numbers in the legends) with or without the addition of DTBP.

[0031]FIG. 6 illustrates the effect of varying the histone/DNA ratio onthe sizes of histone/DNA complexes with the addition of DTBP.

DETAILED DESCRIPTION

[0032] 1. Drug Delivery

[0033] A process for drug delivery is described in which polymerizationand chemical reaction processes take place in the presence of the drugin order to deliver the drug. The polymer is formed from a variety ofmonomers in the presence of the drug and then the mixture is delivered.The mixture could undergo further purification or preparative methods.Drug delivery encompasses the delivery of a biologically active compoundto a cell. This can be accomplished with prokaryotic or eukaryotic cell.It includes mammalian cells that are either outside or within anorganism. It also includes the administration of the drug to the wholeorganism by standard routes such as intravenous, intra-arterial,intra-bile duct, intramuscular, subcutaneous, intraperitoneal, or directinjections into tissues such as the liver, brain, kidneys, heart, eyes,lymph nodes, bone, gastrointestinal tract. It also includes deliveryinto vessels such as blood, lymphatic, biliary, renal, or brainventricles.

[0034] In one preferred embodiment, this process is used to delivernucleic acids. The process of delivering nucleic acids means exposingthe cell to the polynucleic in the presence of the delivery system.Cells indicate both prokaryotes and eukaryotes. The cell is located in aliving organism and exposing is accomplished by administering thenucleic acid and the delivery system to the organism. It also meansmixing the nucleic acids with cells in culture or administering thenucleic acids to a whole organism. Delivering nucleic acids encompassestransfecting a cell with a nucleic acid. These delivery processesinclude standard injection methods such as intramuscular, subcutaneous,intraperitoneal, intravenous, and intra-arterial. It also includesinjections into any vessel such as the bile duct and injections into anytissue such as liver, kidney, brain, thymus, heart, eye, or skin.

[0035] Drugs, pharmaceuticals, proteins, peptides and nucleic acids arebiologically active compounds. The drug can be either the templatepolymer or the daughter polymer. In the preferred embodiment, thetemplate polymer is a polyion, a macromolecule carrying a string ofcharges, such as a nucleic acid which would be termed a polyanionbecause of its average negative charge. The term “nucleic acid” is aterm of art that refers to a string of at least two base-sugar-phosphatecombinations. Nucleotides are the monomeric units of nucleic acidpolymers. The term includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA) in the form of an oligonucleotide, messenger RNA, anti-sense,plasmid DNA, parts of a plasmid DNA or genetic material derived from avirus. The term “nucleic acid” includes both oligonucleic acids andpolynucleic acids. Polynucleic acids are distinguished from oligonucleicacid by containing more than 120 monomeric units. In the case of thetransfer of nucleic acids into cells, the nucleic acid is the template.

[0036] The nucleic acid (polynucleotide) could also be used to produce achange in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic purposes is commonlycalled “gene therapy”. The delivered polynucleotide could produce atherapeutic protein such as a hormone, cytokine, or growth factor. Forexample, the polynucleotide in the form of a plasmid DNA could producethe human growth hormone. The polynucleotide could produce an enzymethat is deficient or defective in patients with an inborn error ofmetabolism. For example, a plasmid DNA could produce phenylalaninehydroxylase which would be therapeutic in patients with phenylketonuria.Furthermore, the polynucleotide could supply an anti-sense that would betherapeutic in patients with a tumor, cancer, or infection. For example,the polynucleotide could be a DNA that is transcribed into an anti-sensemolecule.

[0037] 2. Formation of Polymers

[0038] A polymer is a molecule built up by repetitive bonding togetherof smaller units called monomers. In this application the term polymerincludes both oligomers which have two to ˜80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

[0039] Associated with the polymer in a preferred embodiment is a stericstabilizer which is a long chain hydrophilic group that preventsaggregation of final polymer by sterically hindering particle toparticle electrostatic interactions. Examples include: alkyl groups, PEGchains, polysaccharides, hydrogen molecules, alkyl amines.

[0040] To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer. “Most step-reactionpolymerizations are condensation processes and most chain-reactionpolymerizations are addition processes” (M. P. Stevens PolymerChemistry: An Introduction New York Oxford University Press 1990).

[0041] 2A. Step Polymerization

[0042] In step polymerization, the polymerization occurs in a stepwisefashion. Polymer growth occurs by reaction between monomers, oligomersand polymers. No initiator is needed since there is the same reactionthroughout and there is no termination step so that the end groups arestill reactive. The polymerization rate decreases as the functionalgroups are consumed.

[0043] Typically, step polymerization is done either of two differentways. One way, the monomer has both reactive functional groups (A and B)in the same molecule so that

A-B yields -[A-B]-

[0044] Or the other approach is to have two difunctional monomers.

A-A+B-B yields -[A-A-B-B]-

[0045] Generally, these reactions can involve acylation or alkylation.Acylation is defined as the introduction of an acyl group (—COR) onto amolecule. Alkylation is defined as the introduction of an alkyl grouponto a molecule.

[0046] If functional group A is an amine then B can be (but notrestricted to) an isothiocyanate, isocyanate, acyl azide,N-hydroxysuccinimide, sulfonyl chloride, aldehyde (includingformaldehyde and glutaraldehyde), epoxide, carbonate, imidoester,carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) orsuccinic anhyride. In other terms when function A is an amine thenfunction B can be acylating or alkylating agent.

[0047] If functional group A is a sulfhydryl then function B can be (butnot restricted to) an iodoacetyl derivative, maleimide, aziridinederivative, acryloyl derivative, fluorobenzene derivatives, or disulfidederivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoicacid{TNB} derivatives).

[0048] If functional group A is carboxylate then function B can be (butnot restricted to) a diazoacetate or an amine in whichcarbonyldiimidazole or carbodiimide is used.

[0049] If functional group A is an hydroxyl then function B can be (butnot restricted to) an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or carbodiimide or N, N′-disuccinimidyl carbonate,or N-hydroxysuccinimidyl chloroformate is used.

[0050] If functional group A is an aldehyde or ketone then function Bcan be (but not restricted to) an hydrazine, hydrazide derivative,aldehyde (to form a Schiff Base that may or may not be reduced byreducing agents such as NaCNBH₃).

[0051] Yet another approach is to have one difunctional monomer so that

A-A plus another agent yields -[A-A]-.

[0052] If function A is a sulfhydryl group then it can be converted todisulfide bonds by oxidizing agents such as iodine (I₂) or NaIO₄ (sodiumperiodate), or oxygen (O₂). Function A can also be an amine that isconverted to a sulfhydryl group by reaction with 2-Iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid{TNB} derivatives) can also be used tocatalyze disulfide bond formation.

[0053] Functional group A or B in any of the above examples could alsobe a photoreactive group such as aryl azides, halogenated aryl azides,diazo, benzophenones, alkynes or diazirine derivatives.

[0054] Reactions of the amine, sulfhydryl, carboxylate groups yieldchemical bonds that are described as amide, amidine, disulfide, ethers,esters, isothiourea, isourea, sulfonamide, carbamate, carbon-nitrogendouble bond (enamine or imine) alkylamine bond (secondary amine),carbon-nitrogen single bonds in which the carbon contains a hydroxylgroup, thio-ether, diol, hydrazone, diazo, or sulfone.

[0055] 2B. Chain Polymerization

[0056] In chain-reaction polymerization growth of the polymer occurs bysuccessive addition of monomer units to limited number of growingchains. The initiation and propagation mechanisms are different andthere is usually a chain-terminating step. The polymerization rateremains constant until the monomer is depleted.

[0057] Monomers containing vinyl, acrylate, methacrylate, acrylamide,methaacrylamide groups can undergo chain reaction which can be radical,anionic, or cationic. Chain polymerization can also be accomplished bycycle or ring opening polymerization. Several different types of freeradical initiatiors could be used that include peroxides, hydroxyperoxides, and azo compounds such as 2,2′-Azobis(-amidinopropane)dihydrochloride (AAP).

[0058] 3. Types of Monomers

[0059] A wide variety of monomers can be used in the polymerizationprocesses. These include positive charged organic monomers such asamines, imidine, guanidine, imine, hydroxylamine, hydrozyine,heterocycles (like imidazole, pyridine, morpholine, pyrimidine, orpyrene. The amines could be pH-sensitive in that the pKa of the amine iswithin the physiologic range of 4 to 8. Specific amines includespermine, spermidine, N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD),and 3,3′-Diamino-N,N-dimethyldipropylammonium bromide (Compound 9).

[0060] 1. Monomers can also be oligopeptides, polypeptides or proteins(produced synthetically or in an organism). These oligopeptides can be aNLS peptide which corresponds to the 12 amino acid nuclear localizingsequence of SV40 T antigen, fusion peptides (derived from viruses),endosomolytic peptides and amphipathic peptides. Amphipathic compoundshave both hydrophilic (water-soluble) and hydrophobic (water-insoluble)parts. The amphipathic compound can be cationic or incorporated into aliposome that is positively-charged (cationic) or non-cationic whichmeans neutral, or negatively-charged (anionic). Proteins such as histoneHI can be used. Proteins that bind DNA at sequence-specific sequencessuch as Gal4 protein could also be used.

[0061] Monomers can also be hydrophobic, hydrophilic or amphipathic.Examples of amphipathic compounds include but are not restricted to3,3′-diamine-N-(7-octene)-N-methyldipropylammonium bromide (Compound 7),N,N′-Dinonacrylate-N,N,N′,N′-tetramethylpropanediammonium bromide(Compound 10),N,N′,N″-Trinonacrylate-N,N,N′,N′,N″-pentamethyldiethylentriammoniumbromide (Compound 11) and amphipathic peptides such asCKLLKKLLKLWKKLLKKLKC.

[0062] Monomers can also be intercalating agents such as acridine,thiazole organge, or ethidium bromide.

[0063] 4. Other Components of the Monomers and Polymers

[0064] The polymers have other groups that increase their utility. Thesegroups can be incorporated into monomers prior to polymer formation ofattached to the polymer after its formation. These groups include:

[0065] a. Targeting Groups

[0066] These groups are used for targeting the polymer-drug orpolymer-nucleic acid complexes to specific cells or tissues. Examples ofsuch targeting agents include agents that target to theasialoglycoprotein receptor by using asiologlycoproteins or galactoseresidues. Other proteins such as insulin, EGF, or transferrin can beused for targeting. Peptides that include the RGD sequence can be usedto target many cells. Chemical groups that react with sulfhydryl ordisulfide groups on cells can also be used to target many types ofcells. Folate and other vitamins can also be used for targeting. Othertargeting groups include molecules that interact with membranes such asfatty acids, cholesterol, dansyl compounds, and amphotericinderivatives.

[0067] After interaction of the supramolecular complexes with the cell,other targeting groups can be used to increase the delivery of the drugor nucleic acid to certain parts of the cell. For example, agents can beused to disrupt endosomes and a nuclear localizing signal (NLS) can beused to target the nucleus.

[0068] A variety of ligands have been used to target drugs and genes tocells and to specific cellular receptors. The ligand may seek a targetwithin the cell membrane, on the cell membrane or near a cell. Bindingof ligands to receptors typically initiates endocytosis. Ligands couldalso be used for DNA delivery that bind to receptors that are notendocytosed. For example peptides containing RGD peptide sequence thatbind integrin receptor could be used. In addition viral proteins couldbe used to bind cells. Lipids and steroids could be used to directlyinsert into cellular membranes.

[0069] b. Reporter Groups

[0070] Reporter or marker groups are molecules that can be easilydetected. Typically they are fluorescent compounds such as fluorescein,rhodamine, texas red, cy 5, or dansyl compounds. They can be moleculesthat can be detected by UV or visible spectroscopy or by antibodyinteractions or by electron spin resonance. Biotin is another reportermolecule that can be detected by labeled avidin. Biotin could also beused to attach targeting groups.

[0071] c. Cleavable Groups

[0072] The polymers can contain cleavable groups within the templatebinding part or between the template binding part and the targeting orreporter molecules. When within the template binding part, breakage ofthe cleavable groups leads to reduced interaction of the template anddaughter polymers. When attached to the targeting group, cleavage leadsto reduce interaction between the template and the receptor for thetargeting group. Cleavable groups include but are not restricted todisulfide bonds, diols, diazo bonds, ester bonds and sulfone bonds.

[0073] 5. Template Polymerization

[0074] Template polymerization has been defined as the following (van deGrampel, H. T., Tan, Y. Y. and Challa, G. Macromolecules 23, 5209-5216,1990):

[0075] “Template polymerizations can be defined as polymerizations inwhich polymer chains are able to grow along template macromolecules forthe greater part of their lifetime. Such a mode of propagation can beachieved through the existence of cooperative interactions between thegrowing chain and the template chain and usually leads to the formationof an interpolymer complex. In general, a well-chosen template is ableto affect the rate of polymerization as well as the molecular weight andmicrostructure of the formed polymer (daughter polymer). The concepts oftemplate polymerization were described by Ballard and Bamford with thering opening polymerization of the N-carboxyanhydride ofDL-phenylalanine on a polysarcosine template. Since then, many othersystems involving radical and nonradical initiation of vinyl monomershave been studied in which one or more template effects, arising fromthis peculiar propagation mode, were identified. A number ofradical-initiated template polymerizations have been studied, employingwater as solvent”.

[0076] The main features of template polymerization are:

[0077] 1. Complex formation takes place between polymers

[0078] 2. The rate of polymerization increases as the concentration oftemplate increases. (Fujimori, K., (1979) Makromol. Chem. 180, 1743)

[0079] 3. The structure and conformational features of the template arereflected in the corresponding daughter polymer.

[0080] In template polymerization, propagation of new polymer chainoccurs predominantly along the template, a macromolecular chain, throughspecific cooperative interaction. The nature of interaction can beelectrostatic, H-bonding, charge-transfer, and Van der Waals forces incombination with steriochemical matching. The presence of templateusually affects various polymerization characteristics as well as themicrostructure of the polymer formed. The mechanism of templatepolymerization depends on the degree of monomer adsorption. Two extremecases can be discerned: the adsorption equilibrium coefficient formonomer, K_(M)=-• (type 1) and KM=0 (type 2). In type 1 (“zip” reaction)monomer is fully adsorbed onto all template sites and the polymerizationoccurs only on template. As the K_(M) constant becomes smaller, templatepropagation increasingly proceeds via reaction monomers from thesurrounding solution at the expense of reaction with adjacently adsorbedmonomer. When K_(M)=0 (type 2) only non-adsorbed monomer is present andthe template macromolecules are completely solvated by solvents insteadof the monomers. A prerequisite for template propagation under thiscondition is the growing daughter oligomer, created in bulk solution,that then complexes with template. (“pick-up” reaction). The chainlength below which no complexation takes place (critical chain length)is important for magnitude of the template effect. In fact, there is nosharp border between type 1 and type 2 polymerization's.

[0081] Several processes for using template polymerization for drugdelivery are described. The daughter polymer could be the drug. In apreferred embodiment, the template is the drug (defined to includepharmaceuticals, therapeutic agents or biologically active substances).The process of using template polymerization for drug delivery comprisesmixing the template with monomers and having a daughter polymer formingfrom the monomers. The mixture of template polymer and daughter polymeris then administered to a cell by putting the mixture in contact with acell or near a cell. The mixture of template and daughter polymer couldalso be placed in a pharmaceutical formulation and vial for delivery toan animal. The template polymer could be a polyanion such as nucleicacid including DNA, RNA or an antisense sequence. The DNA can produce atherapeutic agent such as a therapeutic protein or anti-sense RNA.

[0082] In a preferred embodiment, targeting groups could be added duringthe initial template polymerization stage or during subsequentpolymerization steps. In addition, after template polymerization,networks or additional networks can be added to the polymer. These couldbe used to cross-link the polymers. For example, the polymer could becross-linked to put the template into a “cage”. Cross-linking is thelinking of two moieties of a polymer to one another using bifunctionalchemical linker. One result is that the polymer, as a network, becomesstronger and more resistant to being dissolved. Covalent linkingbifunctional linkers may be homobifunctional (which involves the samechemical reaction for linking both moieties) or heterobifunctional(involves two different reactions allowing linkage of differentfunctional groups). By cross-linking, a cage may be formed around ornear the polyion creating a complex of polyion and polymer.Cross-linking the polymer protects the polyion from being destroyed byenzymes and other degrading substrates. For example: If the polyion isDNA, the cross-linked or caging polymer protects DNA from DNases.

[0083] In a preferred embodiment, stable caged polyion particles stillbear a net positive charge. However, it is desirable to recharge it soit would interact less with negatively-charged polymers and particles invivo. Recharging is switching the net polyion particle charge to anopposite charge.

[0084] Complexes may be formed and continue to function in a solution ofchangeable tonicity, which means that the solution can be hypotonic,hypertonic or normal tonicity. Hypotonic means any solution which has alower osmotic pressure than another solution (that is, has a lowerconcentration of solutes than another solution). A hypotonic solution isthe opposite of a hypertonic solution. Normal tonicity in the preferredembodiments is the tonicity of human body fluids, specifically blood.

[0085] 6. Homophase and Heterophase Polymerization

[0086] The chemical reaction and polymerization processes can take placein homophase systems in which the monomer and nucleic acid are in thesame solution. This solution can be water, alcohol, chloroform, esters,organic solvents, or polar aprotic solvents such as DMF or DMSO ordioxane. They can be mixtures of aqueous and organic solvents.

[0087] The chemical reaction and polymerization processes can take placein heterophase systems in which the nucleic acid is in one phase and themonomer is in another phase. Such heterophase systems can be “oil inwater” and also “water in oil” where oil is defined as a solvent thathas low solubility in water. This approach could enable the formation ofmicellar-like structures that have the hydrophobic parts of thepolynucleotide in the inside of a vesicle and the hydrophilic parts onthe outside, or vice-versa. The polymerization reaction can be performedin both direct (oil-in-water) and inverse (water-in-oil) emulsions. Thisapproach allows the use of hydrophobic or amphipathic monomers(Blackley, D. C. Emulsion Polymerization, London: Appl. Sci., 1975).Heterophase polymerization enables vesicles, particles, orsupramolecular complexes to be produced in which the nucleic acid is onthe surface of polymer micelles or the nucleic acid is inside ofmonolayer inverse polymer micelles. In the last case different lipidscan be used for external layer formation. Inverse phase emulsion can beprepared so that in average only one molecules of biopolymer will bepresent in every water drop.(Martinek, K., Levashov, A. V., Klyachko,N., Khmelnitski, Y. L., & Berezin, I. V. (1986) Eur. J. Biochem. 155,453-468).

[0088] 7. Supramolecular Complexes

[0089] A supramolecular complex is a structure that contains two or moredifferent molecules that are not covalently bound. Supramolecularcomplexes can be used for drug delivery and for other purposes such asfor preparative or analytical methods or the construction of newmaterials. We describe a new method for forming a supramolecular complexcontaining nucleic acid and a polymer in which the polymer is formed inthe presence of the nucleic acid. The supramolecular complex can containother components in addition to the nucleic acid and polymer. It cancontain another polymer that is already formed. This already formedpolymer can bind the nucleic acid or the daughter polymer. Theadditional component can be a protein. This protein can be cationic andcontain positive charges that enables it to bind nucleic acid. Suchcationic proteins could be histone, polylysine, or protamine. Thesupramolecular complex could also contain targeting groups.

[0090] A supramolecular complex formed in this fashion could containamphipathic compounds that could be part of liposomes, micelles, orinverse micelles. Liposomes are microscopic vesicles that containamphipathic molecules that contain both hydrophobic and hydrophilicdomains. Liposomes can contain an aqueous volume that is entirelyenclosed by a membrane composed of lipid molecules (usuallyphospholipids) (R. C. New, p. 1, chapter 1, “Introduction” in Liposomes:A Practical Approach, ed. R. C. New IRL Press at Oxford UniversityPress, Oxford 1990). Micelles and inverse micelles are microscopicvesicles that contain amphipathic molecules but do not contain anaqueous volume that is entirely enclosed by a membrane. In micelles thehydrophilic part of the amphipathic compound is on the outside (on thesurface of the vesicle) whereas in inverse micelles the hydrophobic partof the amphipathic compound is on the outside.

[0091] 8. Condensed Nucleic Acids

[0092] A method of condensing nucleic acid is defined as decreasing thelinear length of the nucleic acid. Condensing nucleic acid also meanscompacting nucleic acid. Condensing nucleic acid also means decreasingthe volume which the nucleic acid molecule occupies. A example ofcondensing nucleic acid is the condensation of DNA that occurs in cells.The DNA from a human cell is approximately one meter in length but iscondensed to fit in a cell nucleus that has a diameter of approximately10 microns. The cells condense (or compacts) DNA by a series ofpackaging mechanisms involving the histones and other chromosomalproteins to form nucleosomes and chromatin. The DNA within thesestructures are rendered partially resistant to nuclease (DNase) action.The condensed structures can also be seen on electron microscopy.

[0093] The process of condensing nucleic acid can be used fortransferring nucleic acids into cells or an organism such as for drugdelivery. It could also be used for prepartive or analytical methods orthe construction of new materials.

[0094] We describe a new method for forming condensed nucleic acid byhaving a chemical reaction take place in the presence of the nucleicacid. A chemical reaction is defined as a molecular change in theparticipant atoms or molecules involved in the reaction. An example of amolecular change would be the breaking and forming of covalent bonds ofparticipant compounds. Covalent bonds are defined as havingshared-electron bonds such as those found in carbon-carbon,carbon-nitrogen, carbon hydrogen, carbon-oxygen, carbon-sulfur,carbon-halogen, nitrogen-hydrogen, oxygen-hydrogen, oxygen-oxygen andsulfur-oxygen bonds. The chemical reaction(s) could result in a polymerbeing formed. The polymerization process could take place by the processof template polymerization. A supramolecular complex could form as aresult of this process.

[0095] In a preferred embodiment, one method utilizes covalentlyattaching compounds to polyions such as genes for enhancing the cellulartransport of the polyion while maintaining its functionality. A patentapplication entitled: A Method For Covalent Attachment Of Compounds ToGenes, Serial No. ______, filed Dec. 12, 1997 describing methods ofcovalently attaching compounds as well as structures used therein isincorporate herein by reference. Although the cited application teachesthe attachment of compounds to genes, the methods and structures may beapplied to attaching molecules to a polymer as discussed in the presentspecification and the term polymer is not limited to nucleic acids.

[0096] Signals that enhance release from intracellular compartments(releasing signals) can cause polyion release from intracellularcompartments such as endosomes (early and late), lysosomes, phagosomes,vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network(TGN), and sarcoplasmic reticulum. Release includes movement out of anintracellular compartment into cytoplasm or into an organelle such asthe nucleus. Releasing signals include chemicals such as chloroquine,bafilomycin or Brefeldin At and the ER-retainingsignal (KDEL sequence),viral components such as influenza virus hemagglutinin subunit HA-2peptides and other types of amphipathic peptides.

[0097] Nuclear localizing signals enhance the entry of a polyion intothe nucleus or directs the gene into the proximity of the nucleus. Suchnuclear transport signals can be a protein or a peptide such as the SV40large T ag NLS or the nucleoplasmin NLS.

[0098] 9. A Method for Forming a Polymer that Binds Nucleic Acids

[0099] We describe a process of forming in the presence of the nucleicacid a polymer that has affinity to nucleic acid. This excludes theprocess of forming polymers that are proteins or nucleic acids. It alsoexcludes polymers that bind the nucleic acid by Watson-Crick binding.Watson-Crick binding is defined as the normal base-pairing arrangementin which guanine base pairs with cytosine base and in which theadenosine base pairs with thymine bases. Affinity indicates that thepolymer is attracted to nucleic acid and remains bound to it bynon-covalent forces (such as van der waal, hydrogen bonds, and ionicbonds) under either physiologic or non-physiologic conditions.

[0100] The process of forming a polymer in the presence of the nucleicacid can be used for transferring nucleic acids into cells or anorganism such as for drug delivery. It could also be used forpreparative or analytical methods or the construction of new materials.

[0101] The nucleic acid-binding polymer can form as a result of templatepolymerization.

[0102] We also describe a process of forming an interpolyelectrolytecomplex containing nucleic acids by having a chemical reaction takeplace in the presence of the nucleic acid. An interpolyelectrolytecomplex is defined as a mixture of two polymers with opposite charges.In this situation the nucleic acid is a polyanion and the formed polymeris a polycation.

[0103] Definitions of Compounds Used in Preferred Embodiments

[0104] Orthogonal—Refers to a protective (protecting) group that can beselectively removed in the presence of other protective groups containedon the molecule of interest.

[0105] Monovalent—refers to an ionic species possessing 1 charge.

[0106] Protective Group—A chemical group that is temporarily bound tofunctionalities within a multifunctional compound that allows selectivereactions to take place at other sites within the compound. Commonprotective groups include, but are not limited to carbamates, amides,and N-alkyl groups.

[0107] Functionality—Refers to general classes of organic compounds suchas: alcohols, amines, carbonyls, carboxyls, and thiols.

EXAMPLES

[0108] Overview of Experimental Design

[0109] The following examples show that polymerization can take place inthe presence of DNA. Since the central feature of these polymers istheir ability to bind DNA, we selected a relatively simple assay todetect the formation of such polymers and that is agarose gelelectrophoresis with ethidium bromide staining of DNA. A strongDNA-binding polymer retards (or slows) the migration of the DNA in thegel. In the experimental samples where the DNA is already present duringthe polymerization (reaction) process, the sample is simply loaded ontothe agarose gel. In the control samples where DNA is not present duringthe reaction process, the DNA is added after the reaction. This approachis also a powerful method to determine whether any polymer is formed bya template polymerization process. That is, if the polymer only formswhen the template DNA is present and not when the template DNA is absentthen this is definitive proof of template polymerization. The initialresults with agarose gel electrophoresis are followed up with moresophisticated assay for polymers and particles that include gelfiltration (size exclusion) chromatography, transmission electronmicroscopy, and particle sizing by dynamic light scattering.

[0110] The process of polymerization in the presence of nucleic acidscan be used to transfer and express genes in cells. Besides showing theutility of this process, it also indicates that the chemical reactionswere not chemically modifying or destroying the nucleic acid. A anotherapproach was also used to detect nucleic acid damage. We incorporateddisulfide bonds into the polymers and then broke the polymers down byadding dithiothreitol (DTT also known as Cleland's reagent) whichreduces the disulfide bonds. After the breakdown of the polymers thenucleic acid, DNA (that was within the polymer particles) wastransfected into cells using another transfection method (with acationic lipid). Expression was the same as the native DNA. Expressionis a very sensitive indicator of any destruction or modification alongthe entire length of the reporter (luciferase) gene and promoter. Thesepolymers were designed with disulfide bonds so that they could moreeasily be broken down inside cells.

Example 1

[0111] Step polymerization with DNA as a template was performed usingthe polyamine N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD) anddithiobis(succinimidylpropionate) (DSP). This template polymerizationwas done using two different monomer species together in which each ofthe species possessed at least two reactive ends to propagate a growingchain. Using a bifunctional amine with affinity to plasmid DNA as amonomer and bifunctional aminoreactive cross-linker as a co-monomer, wedemonstrated that 1) it is possible to obtain DNA-bound polyamide as aresult of such polymerization, and 2) the resulting polymer can condensetemplate DNA into compact structures.

[0112] Methods:

[0113] The following amine was used as monomers:

[0114] 1. N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Aldrich,Milwaukee, Wis.)

[0115] The following cross-linker was used as a co-monomer:

[0116] 1. Dithiobis(succinimidylpropionate), (DSP, S-S cleavable bissuccinimide ester, Pierce, Rockford, Ill.)

[0117] Optimized reaction conditions with AEPD/DSP were as follows.Plasmid DNA (pCIluc, 50 mg) and AEPD (10 mg) were mixed in 50 ml ofbuffer solution (0.1 M HEPES, 1 mM EDTA, pH 7.4). After 5 min DSP (60 mgin 1.5 ml of dimethylformamide) was added. After mixing, the reactionwas left for 1 hour in the dark at room temperature. Finally, reactionmixture was dialysed against water or desired buffer solution inmicrodialysis cell with a molecular weight cut-off of 1,000 (Rainin,Ridgefield, N.J.).

[0118] The pCILuc plasmid expresses a cytoplasmic luciferase from thehuman immediately early cytomegaloviral (CMV) promoter. It wasconstructed by inserting the cytoplasmic luciferase cDNA into the pCI(Promega Corp., Madison, Wis.) CMV expression vector. Specifically, aNihau/EcoRI restriction digestion fragment containing the cytoplasmicluciferase cDNA was obtained from pSPLuc (Promega Corp.) and insertedinto pCI pDNA that was digested with NheI and EcoRI. Plasmid DNA waspurified using the Qiagen (Chatsworth, Calif.) plasmid purificationsystem (alkaline lysis followed by anion exchange chromatography).

[0119] Agarose gel electrophoresis and ethidium bromide staining of theDNA was done using standard techniques (Sambrook, J., Fritsch, E. F.,and Maniatis, T. (1989) in Molecular Cloning Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

[0120] Standard gel filtration (size-exclusion) chromatography wasperformed to determine the size of the polymers that formed in thepresence and absence of DNA. Since the DNA strongly bound the polymer,it was necessary to first remove the DNA. This was accomplished byvigorous DNase digestion. Samples of DNA/AEPD/DSP reaction mixture (50ug total DNA, pCIluc) were supplemented with 5M NaCl solution up to 0.5M NaCl. DNase I (Sigma) was added to the mixture (0.06 U/ug DNA). DNasedigestion was carried out in the buffer containing 10 mM Tris, 10 mMMgCl2, 1 mM CaCl2, pH 7.0, for 4 hrs at 37° C. After this reaction, themixture was centrifuged at 12,000 rpm for min and applied on theSephadex G-75 (Sigma) column (0.8×20 cm) equilibrated with mM HEPES, 0.5NaCl, pH 7.4. Fractions (0.5 ml) were analyzed for OD 260.

[0121] Transmission electron microscopy of the formed complexes usingstandard negative staining procedures on coated grids. After the sampleswere stained with methylamine tungstate (BioRAD), the grids wereexamined using a Jeol 100CX transmission electron microscope.

[0122] The preparation for light scattering was prepared essentiallywith the same DNA/AEPD/DSP ratios as for EM (see optimized AEPD/DSP) butwith 3 mg of DNA (pCIluc). DNA/AEPD mixture was incubated withoccasional vortexing for 10 min at room temperature before addition ofDSP. The sample was centrifuged at 12,000 rpm for min and passed through0.2 um polycarbonate filter (Poretics Corp., Livermore, Calif.) andanalyzed using Particle Size Analyser equipped with 15 M argon laser(Brookhaven Instruments, Inc.).

[0123] Results:

[0124] Agarose gel electrophoresis of the final experimental complexes(formed by reacting AEPD and DSP in the presence of DNA) demonstratedcharacteristic gel retardation of plasmid DNA in the gel in which thecomplexed plasmid DNA migrated more slowly than the original plasmidDNA. In addition there was some DNA material in the starting well. Thefinal complexes were also treated with 25 mM dithiothreithol (DTT)(Fisher, Itasca Ill.) for 30 minutes at 37° C. to cleave the disulfidebonds within the polymer (part of the DSP co-monomer). The DTT treatmentreversed the electrophoretic pattern back to that of the native plasmidDNA and no retarded DNA material was present. This indicates that theretarded pattern was due to the polymer complexing with the DNA. It alsoindicates that the monomers or polymer did not react with the DNA.Transfection of the DNA (after DTT treatment) into cells in cultureusing a commercial transfection reagent (LT-1, Mirus, Madison, Wis.)resulted in as much luciferase expression as native DNA. This alsoindicates that the DNA was not chemically modified.

[0125] A control sample contained AEPD and DSP at the sameconcentrations but plasmid DNA was omitted during the reaction. The DNAwas added after the reaction was completed. Agarose gel electrophoresisshowed much less retardation of the DNA than the above experimentalsample. This indicates that polymerization did not occur in the controlsample and that the polymerization observed in the experimental sampleoccurred by template polymerization.

[0126] Further studies were performed to determine the size of thepolymer that formed in the presence of the DNA. This was accomplished byfirst digesting the DNA exhaustively with DNase and then running theremaining polymer through a size-exclusion column. Gel filtration of thecomplex's exhaustive DNase lysate in 0.5 M NaCl demonstrated formationof the product with apparent molecular weight of >3,000 Da. The controlsample (DNA added after reaction of AEPD and DSP) did not contain anylarge polymer of this molecular weight. This indicates that the polymerthat formed in the presence of DNA occurred by template polymerization.

[0127] Physical methods were employed to determine directly the size andshape of the polymer/DNA complexes. Transmission electron microscopy ofthe experimental complexes (formed by reacting AEPD and DSP in thepresence of DNA) revealed formation of spherical structures with 40-50nm in diameter (individual and aggregated) (FIG. 2). Dynamic lightscattering of the same preparation yielded average particle size of 80nm. These results are consistent with the ability for the particles topass through 0.2 micron filters. The control samples (DNA added afterreaction of AEPD and DSP) did not contain any particles on electronmicroscopy or particle sizing.

[0128] Findings:

[0129] 1. The polyamine was co-polymerized with DSP to form a polymer inthe presence of DNA and this polymer was bound to the DNA.

[0130] 2. The polymer formed by a process of template polymerization.

[0131] 3. The polymer condensed the DNA to form particles less than 80nm in diameter.

[0132] 4. The DNA was not chemically modified by the polymerizationprocess and was still able to express luciferase after transfection intocells in culture.

EXAMPLE 2

[0133] Step polymerization with DNA as a template was performed usingthe polyamine N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD) as inExample 1 above except DPBP was used as the co-monomer.

[0134] Methods:

[0135] The following amine was used as monomers:

[0136] 1. N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Aldrich,Milwaukee, Wis.)

[0137] The following cross-linker was used as a co-monomer:

[0138] 1. Dimethyl-3,3′-dithiopbispropionimidate (DPBP, S-S cleavablebisimido ester, Pierce)

[0139] Optimized reaction conditions with AEPD/DPBP were as follows.Plasmid DNA (pCIluc, 50 mg) and AEPD (24 mg) were mixed in 150 ml ofbuffer solution (20 mM HEPES, 1 mM EDTA, pH 7.4). After 5 min DPBP (155mg in 5 ml of methanol) was added. After mixing, the reaction was leftfor 1 hour in the dark at room temperature.

[0140] Results:

[0141] Unlike the bissuccinimidate reaction (example 1), diimidoestercross-linking (used in this example) preserves positive charges ofaminogroups by converting them into amidines. Therefore, extremelypositively charged polymer was formed as a result of this reaction whichcaused complete DNA retardation on agarose gels. DNA addition to thereaction mixture after the reaction between amine and cross-linker didnot induce DNA retardation on the gel. Treatment of retarded complexeswith DTT resulted in restoration of the native plasmid electrophoreticpattern.

[0142] Findings:

[0143] 1. Step polymerization of AEPD and DPBP occurred in the presenceof DNA and resulted in a polymer that bound DNA very strongly.

[0144] 2. The polymer formed by template polymerization.

[0145] 3. The DNA was not chemically modified and could be recoveredintact after DTT treatment.

EXAMPLE 3

[0146] Step polymerization with DNA as a template was performed usingthe polyamine N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD) as inExample 1 above except 2-iminothiolane (Traut's reagent) was used as theco-monomer. This is an example of ring opening of the 2-iminothilane andthen oxidation of SH groups that form as a result of the ring opening.

[0147] Methods:

[0148] The following amine was used as monomers:

[0149] 1. N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Aldrich,Milwaukee, Wis.)

[0150] The following cross-linker was used as a co-monomer:

[0151] 1. 2-iminothiolane (thiol/amino-linking heterobifunctional agent,Pierce) Optimized reaction conditions with AEPD/2-iminothiolane were asfollows. Plasmid DNA (pCILuc, 50 mg), AEPD (1 mM) and iminothiolane (4mM) were mixed in 450 ml of buffer solution (20 mM HEPES, 1 mM EDTA, pH8.0). After 30 min 5 ml of iodine solution (40 mM in ethanol) wereadded. Reaction was allowed to stand for 1.5 h in the dark at roomtemperature.

[0152] Results:

[0153] Generally, the above procedure is two-step polymerization withreactive monomer formation. 2-iminothiolane forms bisthiol AEPDderivative on the DNA which can be further polymerized by oxidizing SHgroups with molecular iodine. Results for DNA gel retardation and DTTtreatment are basically the same as for AEPD/DPBP pair. Under conditionsindicated above DNA and AEPD/2-iminothiolane polymer form truly solublecomplex completely retarded in agarose gel.

[0154] The DNA in the control sample (DNA added after the polymerreaction) was not retarded on gel electrophoresis.

[0155] Findings:

[0156] 1. Ring opening and two-step polymerization processes can be usedfor forming template polymers that bind to DNA.

[0157] 2. Polyamines can be polymerized in the presence of DNA using theconversion amines to SH groups with subsequent oxidation reactions.

EXAMPLE 4

[0158] Similar results were obtained when spermine was used instead ofAEPD as in Example 1. Plasmid DNA (10 ug) and spermine (1.5 ug) weremixed in 15 ul of 0.1M HEPES, pH 8.0. After 5 min of incubation DSP (25ug in 1 ml of DMF) was added with intensive mixing. After 1 hrincubation at room temperature DNA was analyzed on agarose gel. In caseof “DNA after” experiment, DNA (10 ug) was added after quenching DSPreaction with 0.1 M glycine for 0.5 hr. Electrophoretic pattern wasfound similar to the one with AEPD/DSP in Example 1.

EXAMPLE 5

[0159] A novel amine was used as a monomer in conjunction with DTBP fortemplate polymerization of DNA.

[0160] Methods:

[0161] The following amine was used as a monomer:

[0162] 1.3,3′-(N′,N″-tert-butoxycarbonyl)-N-(7-octene)-N-methyldipropyl-ammoniumbromide (compound 7, see synthesis section).

[0163] Following cross-linkers were used as co-monomers:

[0164] 1. Dimethyl-3,3′-dithiopbispropionimidate (DTBP, S-S cleavablebisimido ester, Pierce) Optimized reaction conditions with compound6/DTBP were as follows. Plasmid DNA (pCILacZ, 10 mL of a 3.4 mg/mL stocksolution, 34 mg, 103 nmol nucleotide base) and compound 6 (3 mL of a1.29 mg/mL stock solution, 39 mg, 108 nmol) were mixed with 85 ml waterand 10 ml of buffer solution (0.2 M HEPES, 10 mM EDTA, pH 8.0). DTBP(1.1 mL of a 100 mM solution in DMF, 33.7 mg, 109 nmol) was added. Aftermixing, the reaction was left for 1 h in the dark at room temperature.

[0165] The pCILacZ plasmid was similarly constructed by placing therestriction digestion fragment of the E. coli β-galactosidase gene intothe pCI vector.

[0166] Results

[0167] Agarose gel electrophoresis of the final complexes demonstratedcharacteristic retardation of the plasmid DNA. The control sample (DNAadded after reaction) did not show any retardation.

EXAMPLE 6

[0168] A peptide was used as a monomer for polymerization in thepresence of DNA and this process enable the formation of complexes thatexpressed luciferase after transfection into cells in culture.

[0169] Methods:

[0170] NLS peptide corresponds to the 12 amino acid nuclear localizingsequence of SV40 T antigen. This peptide was synthesized by GenosysBiotechnologies Inc with a Cysteine on each end for cross-linkingpurposes (MW=1481) Histone Hi was obtained from Sigma. The cross-linkersDSP (dithiobis[succinimidylpropionate]) and DPDPB(1,4-Di-[3′-(2′-pyridyldthio)-propionamido)]butane) were purchased fromPierce.

[0171] The NLS peptide was mixed with plasmid DNA (pCILuc) at variousratios (0.4, 0.8, 1.2, 1.6) in 20 mM HEPES pH 7.5, 1 mM EDTA at aconcentration of plasmid DNA of 0.3 mg/ml. The disulfide cleavablecross-linker DPDPB was added to final concentrations of 2 and 6 mM andthe mixtures were incubated for 30 minutes at room temperature in thedark. Reaction products were analyzed by agarose gel electrophoresis andDNA was visualized by ethidium bromide staining. Extent ofpolymerization of cationic monomers (NLS peptides) was determined onSDS-PAGE. Briefly, NLS peptide/pDNA complexes (with and without DPDPBcross-linker) were incubated with 2.5 units DNase I for 1 hour at 37° C.to remove the DNA from the complexes. Following digestion, remainingprotein components were run on a 15% SDS-PAGE and stained with coomassieblue.

[0172] All transfections were performed in 35 mm wells using 2 ug pDNAper well. NLS peptide/pDNA complexes (with and without DPDPBcross-linker) were diluted in Opti-MEM (Life Technologies) and afusogenic cationic polyamine (ODAP, Mirus Corp, Madison, Wis.) was addedto enhance transfection. It is believed that this polyamine helpsfacilitate intracellular endosomal escape of the complexes into thecytoplasm. Pre-formed complexes were incubated with phosphate bufferedsaline washed NIH3T3 cells for 4 hours at 37° C. Transfection complexeswere then removed and replaced with complete growth medium. Cells weregrown for 40-48 hours and harvested and assayed for reporter geneexpression (luciferase).

[0173] For determination of luciferase activity, cells were lysed by theaddition of 100 ul for 25 mm-in-diameter plates and 200 ul for 35mm-in-diameter plates of lysis buffer (0.1% Triton X-100, 0.1MK-phosphate, 1 mM DTT pH 7.8). 20 ul of the cellular extract wereanalyzed for luciferase activity as previously reported (Wolff, J. A.,Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465-1468, 1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)luminometer was used. Results:

[0174] The stepwise cross-linking of NLS peptides along the DNA templatedrastically alters the mobility of pDNA in agarose gel electrophoresis.At low peptide to pDNA ratios (0.4:1, 0.8:1) the NLS peptide/pDNA/DPDPBcomplexes migrated as a characteristic smear with several prominentbands as compared to NLS peptide/pDNA complexes without cross-linkerwhich migrated similarly to pDNA alone. At higher ratios (1.2:1, 1.6:1)the net charge of the complexes becomes positive and precipitationoccurs with or without the DPDPB cross-linker. When the pDNA is addedafter the polymerization reaction (NLS peptide/DPDPB) the agarose gelmigration pattern looks nearly identical to NLS peptide/pDNA complexeswithout cross-linker indicating that polymerization did not occurwithout the template.

[0175] To determine the degree of polymerization of the NLS peptideswithin the NLS peptide/pDNA/DPDPB complexes, products were analyzed on15% SDS-PAGE (without reducing agents) after DNA removal (by DNase Idigestion) as illustrated in FIG. 3. Multimers of the NLS peptide wereobserved only in the reactions when cross-linker and template (PDNA)were present together with the NLS peptide monomers (panel A, lanes 6and 7). With DTT (50 mM) treatment prior to SDS-PAGE the NLS peptides inthe template polymerized reactions migrated at positions correspondingto monomers once again (panel B, lanes 6 and 7) indicating thatdisulfide bonds were present in the linkages between the monomers.Lanes: M-marker protein standards; 1—NLS peptide alone (7 μg); 2—DNase Ialone (2.5 u); 3—NLS peptide/pDNA; 4—NLS peptide/DPDPB (2 mM)+pDNA addedafter the polymerization reaction; 5—NLS peptide/DPDPB (6 mM)+pDNA addedafter the polymerization reaction; 6—NLS peptide/DPDPB (2 mM)/pDNA;7—NLS peptide/DPDPB (2 mM)/pDNA. Protein staining clearly shows a ladderof increasing size bands indicating a stepwise polymerization of NLSpeptides with dimers appearing as the fastest migrating species. Thisladder of bands was only observed in the reactions when the cross-linker(DPDPB) was present together with the pDNA and the NLS peptideindicating that polymerization proceeded in a template dependentfashion. In addition, treatment of the complexes with the reducing agentDTT in the sample buffer completely abolished the ladder indicating thatthe ladder was a result of NLS peptide cross-linking via the disulfidecontaining DPDPB.

[0176] Polyamine mediated transfections performed with NLSpeptide/pDNA/DPDPB complexes resulted in increased level of luciferaseproduction as compared to transfections with NLS peptide/pDNA alone orNLS peptide/DPDPB polymerization prior to pDNA addition (Table 1). Forexample, with the 0.8:1 ratio of peptide to DNA compare the luciferaselevels in the control sample (9.4 million, second row, NLS+2 mM DPDPB,DNA added after reaction) to the levels in the experimental sample(365.9 million, third row, NLS +2 mM DPDPB in the presence of DNA). Theprocess of template polymerization caused a ˜40-fold increase inexpression. TABLE 1 Luciferase expression (in light units per 35 mmwell). Total Light Units per 35 mm well × 10⁶ 0.4:1 ratio of peptide to0.8:1 ratio of peptide to Condition DNA DNA NLS + 2 mM DPDPB, 4.4 9.4DNA added after reaction NLS + 2 mM DPDPB in 10.9 365.9 the presence ofDNA NLS + 6 mM DPDPB in 5.7 393.1 the presence of DNA

[0177] Findings:

[0178] 1. Peptides can be used for template polymerization in thepresence of DNA.

[0179] 2. This process enables complexes to be prepared that cantransfect mammalian cells efficiently.

EXAMPLE 7

[0180] Chain Polymerization using 1-Vinylimidazole (VIm) as a Monomer,2,2′-Azobis(-amidinopropane)dihydrochloride (AAP) as an Initiator andPlasmid DNA as a Template

[0181] Methods:

[0182] The conditions for template polymerization of 1-vinyl imidazole(VIm) as a monomer using 2,2′-Azobis(-amidinopropane) dihydrochloride(AAP) as an initiator were as follows. A 400 mM stock solution of VIm(TCI America OGBO1, MW 94.72, density 1.04) was prepared with steriledeionized water. The pH was adjusted to 6 with HCl. Then the solutionwas degassed with nitrogen gas. A 200 mM stock solution of AAP (Wako 11G2606, MW 271.2) was also prepared with sterile deionized water anddegassed with nitrogen gas. 20 mM of plasmid DNA (pBlueRSVLux, 800 ul of6.9 mg/ml) was mixed with 20 mM of VIm and 2 mM of AAP from the stocksolutions above. A control sample contained VIm and AAP at the sameconcentrations but plasmid DNA was omitted. Both the experimental(VIm/AAP/DNA) and control (VIm/AAP but no DNA) reactions were performedin sterile deionized water. The reactions were incubated for 2 hours at50° C. and then the samples were analyzed by agarose gel electrophoresisfollowed by ethidium bromide staining. 20 mM of plasmid DNA was added tothe control sample prior to loading it on the gel.

[0183] The previously described, plasmid DNA pBlueRSVLux (also known aspBS.RSVLux) was used to express the firefly luciferase reporter genefrom the Rous Sarcoma Virus (RSV) LTR promoter (Danko, I., Fritz, J. D.,Jiao, S., Hogan, K., Latendresse, J. L., and Wolff, J. A. Gene TherapyPharmacological enhancement of in vivo foreign gene expression inmuscle. volume 1, pp. 114-121, 1994). The plasmid also contains the SV40intron and poly A addition signals for proper and efficient mRNAprocessing. Results

[0184] The agarose gel electrophoresis analysis showed that the plasmidDNA in the control sample (DNA added after the reaction) migrated withthe same pattern as the original plasmid DNA. In the experimental sample(DNA present during the reaction), the plasmid DNA was retarded with DNAbands migrating slower than the original-plasmid DNA. There was also DNAstaining material in the starting wells.

[0185] Findings:

[0186] 1. A polyvinyl imidazole polymer formed in the presence of DNAand this polymer was complexed with the DNA as evident by gelelectrophoresis analysis.

[0187] 2. This polymer formed by template polymerization because thepolymer did not form if the template DNA was omitted.

EXAMPLE 8

[0188] Template Polymerization (Caging) of Large Polymers

[0189] Methods:

[0190] Poly-L-lysine (hydrobromide, molecular mass from 30 to 70 kDa)(PLL) and Polyallylamine (hydrochloride) (55 kDa) (PAA) were obtainedfrom Aldrich. Histone Hl(Type III-S from Calf Thymus) was obtained fromSigma. Dimethyl 3,3′-dithiobispropionimidate(DTBP) was purchased fromPierce. The polycations were dissolved in de ionized water: PLL and H1to concentration 10 mg/ml and PAA to 2 mg/ml. DTBP was dissolved in H2O(30 mg/ml) immediately before utilization.

[0191] DNA/polycation complexes were prepared by the rapid mixing of 37μg of plasmid DNA with varying amounts of polycations in 750 μl of 25 mMHEPES pH 8.0, 0.5 mM EDTA. The mixtures were kept 30 min at roomtemperature and various amounts of DTBP were added. The mixtures wereincubated 2 hours at room temperature. 2M NaCl was added to thecomplexes to final concentration 100 mM while vigorously mixing.

[0192] Ninety degree light scattering measurements were performed usinga Fluorescence Spectrophotometer. The wavelength setting was 600 nm forboth the incident beam and detection of scattering light. The slits forboth beams were fixed at 2 nm. The size of the resulting complex wasdetermined by light scattering on a Brookhaven ZetaPlus particle sizer.The samples were centrifuged at 12,000 g for 7 min. The amount of DNAremaining in the supernatant was determined by measurement of theabsorbency at 260 and 280 nm.

[0193] Results:

[0194] Effect of DNA/PLL Ratio and NaCl on the Light Scattering.

[0195] PLL was added to plasmid DNA in 0.75 ml of 25 mM HEPES pH 8.0while vigorously mixing. The kinetics of light scattering was determinedimmediately after mixing. The turbidity of DNA/PLL complexes was wellabove that of free DNA in all range of PLL concentration As shown inFIG. 4 complex aggregation increased when molar charge ratio PLL/DNA toapproximate to 1 and was maximal at ratio 1.17. Further increases in PLLconcentration resulted in decreasing of complex turbidity. The lightscattering did not change with time for at least for 30 min.

[0196] At low positive to negative charge ratio water-solublenonstochiometrical complexes are formed. At ratio 1 the complexes becomeinsoluble. Increasing the content of polycation may lead to the complexchanging its sign and becoming soluble again. Presumably the particlesare stabilized in solution by the positively charged loops and danglingtails of the polycation bind to the chain DNA. With increasing saltconcentration to 100 mM the charge stabilized complexes (ratio +/− morethen 1) started to aggregate (FIG. 4). The velocity of aggregationdecreased with increasing PLL/DNA ratio, but final turbidity level wasthe same for all samples.

[0197] Effect of DTBP on DNA/PLL Complexes Light Scattering.

[0198] The incubation of DNA/PLL complexes with 0.97 umol of DTBP for 2h at room temperature resulted in a shift of turbidity maximum to aPLL/DNA ratio of 0.88 (FIG. 4). That can be explained as increasing ofPLL charge as a result of modification. Apparently, DTBP did notcrosslink PLL/DNA complexes with each other at ratio more then 1. Theaddition of NaCl to a concentration of 100 mM did not change lightscattering throughout the range of PLL concentration (FIG. 4). Theseresults indicate that the addition of DTBP prevented the PLL/DNAcomplexes from aggregating in 100 mM salt.

[0199] The ability to centrifuge the DNA was used as another indicationof aggregation (Table 2). All samples were centrifuged 7 min at 12,000rpm and the amount of DNA in supernatant was determined. As showncrosslinked PLL/DNA complexes with molar ratio 4.1 and 5.9 did notprecipitate. Therefore the size of complexes were very small. Incontrast, DNA in noncrosslinked complexes were completely precipitated.TABLE 2 The effect of DTBP on the precipitation of plasmid DNA/PPLcomplexes in the presence of 100 mM NaCl. % DNA in solution aftercentrifugation PLL/DNA ratio −DTBP +DTBP 0.585 67  77 0.879 0  0 1.171 0 0 2.342 0 17 4.098 0 97 5.854 0 97

[0200] Effect of PLL/DNA Ratio on the Size of Complexes. TABLE 3 Theeffect of varying the DNA/PLL charge (monomoer:monomer) ratio on thesizes of PLL/DNA complexes with the addition of 0.97 umol DTBP. Thesizes were determined by quasi elastic light scattering and numbersindicate the percent of particles <100 nm or >100 nm. Number inparentheses indicate the size (diameter in nm) of the most abundantspecies within that size range. Percentage of Particles Less or GreaterThan 100 nm no NaCl +100 mM NaCl DNA/PLL <100 nm >100 nm <100 nm >100 nm0.43 72(50) 28(200) 36(28) 64(280) 0.65 68(42) 32(196) 36(63) 64(304)0.88 — 100(10000) — 100(10000) 1.31 — 100(10000) — 100(10000) 1.74 8(65)92(150,680) 7(84) 93(1000) 2.61 69(33) 31(118) 11(91) 89(836) 4.1296(43.4) 4(6580) — 100(204,1152) 6.18 100(22.4) — — 100(222,1052) 0.43 +DTBP 29(55) 71(331) 43(31) 57(131,374) 0.65 + DTBP 43(31) 69(339) 16(54)84(350) 0.88 + DTBP 13(72) 87(431,1640) 21(41) 79(707,4690) 1.31 + DTBP87(45,100) 3(260) — 100(10000) 1.74 + DTBP 87(45,99) 3(256) 73(55)27(191) 2.61 + DTBP 100(32,98) — 77(51) 23(130) 4.12 + DTBP 99(27.9)1(6468) 69(67.6) 31(142,2000) 6.18 + DTBP 94(35.2) 6(6580) 96(68)4(6813) 4.12 + DTBP + — 100(362,8800) DTT 6.18 + DTBP + — 100(381,8755)DTT

[0201] In Table 3, it is clear, that PLL/DNA complexes with ratio higherthen 1.3 became substantially less prone to aggregate in the presence of100 mM NaCl after DTBP modification. The PLL/DNA complex stabilizedreaction is intra complex crosslinking because the treatment of themodified PLL/DNA complexes with ratio 4.12 and 6.18 by 50 mM DTT for 1 hresulted in aggregation. In this condition the crosslinks should becleaved but the level of lysine modification is not changed.

[0202] Effect of PLL/DTBP Ratio on the Size and Stability of PLL/DNAComplexes.

[0203] The PLL/DNA complex in ratio 4.12 was treated by differentconcentrations of DTBP during 2 h. The size of particles without and inpresence of 100 mM NaCl was determined by quasi elastic lightscattering. TABLE 4 The effect of varying the DTBP/PLL ratio (molarratio of DTBP to lysine residue) on the sizes of PLL/DNA complexes. Thesizes were determined by quasi elastic light scattering and numbersindicate the percent of particles <100 nm or >100 nm. Number inparentheses indicate the size (diameter in nm) of the most abundantspecies within that size range. Percentage of Particles Less or GreaterThan 100 nm DTBP/PLL no NaCl + 100 mM NaCl for 1 h Ratio <100 nm >100 nm<100 nm >100 nm 0 75(88) 25(586) — 100(7524) 1.01 93(44) 7(6874) 37(92)63(600) 2.03 95(35) 5(550) 75(66) 25(190,4658) 3.05 100(52) — 100(86) —

[0204] Table 4 shows that an excess of DTBP was needed for complexprotection from salt dependent aggregation. It should be noted that DTBPup to ratio of 3.05 did not induce crosslinking between DNA/PLLparticles. For samples with DTBP/PLL ratio 2.03 and 3.05 zeta potentialwere 16.16±3.23 mV and 20.33±3.3 mV respectively in 25 mM HEPES pH 8.0,100 mM NaCl.

[0205] Stability of DNA/PLL Complexes to Disruption by Polyanion DextranSulfate (DS).

[0206] DNA/PLL complexes (molar ratio of 0.87, 1.74, 3.04 or 4.35 asindicated in FIG. 5) were prepared as before but in 1 ml of buffer. 0.97umol of DTBP were added. The mixtures were incubated 2 hours at roomtemperature. 10 ul of ethidum bromide (EB) (0.1 mg/ml) were added inevery sample and the samples were incubated 30 min. The aliquot portionsof DS were then added sequentially, with mixing. After each addition,the fluorescence was allowed to stabilize 30 seconds.

[0207] Addition of PLL to DNA in solution gave rapid falls influorescence, corresponding to complex formation. Addition of DS topre-formed complexes can restore EB fluorescence and can be taken asindicator of complex stability (FIG. 5). Without DTBP, the EBfluorescence rose with the addition of DS in every ratio of PLL/DNA(FIG. 5). With DTBP, the increase was attenuated and there was a clearinfluence of DTBP modification on complex stability: the fraction ofcomplexes could not be disrupted in any DS concentration. The part ofcomplexes which are stable to disruption by DS depended on PLL/DNAratio.

[0208] DNA/PAA Complexes.

[0209] Polyallylamine (PAA) is similar to PLL and contains primary aminogroups. But average pK of PAA is low then PLL because stronger influenceof one group to another. TABLE 5 The effect of varying the DNA/PAA ratioon the sizes of PAA/DNA complexes with or without the addition of DTBP.The sizes were determined by quasi elastic light scattering and numbersindicate the percent of particles <100 nm or >100 nm. Number inparentheses indicate the size (diameter in nm) of the most abundantspecies within that size range. Percentage of Particles Less or GreaterThan 100 nm no NaCl +NaCl PAA/DNA Ratio <100 nm >100 nm <100 nm >100 nm2.17 + DTBP 7(106) 77(455),16(4436) — 100(2064) 4.34 + DTBP 93(66)7(6900) 70(94) 30(916) 6.51 + DTBP 93(53) 6(163) 81(92) 19(870) 8.68 +DTBP 97(55) 3(5607) 81(62) 19(182) 4.34 55(71) 45(352) 4(79) 96(863)

[0210] The results in Table 5 are very similar to the results withPLL/DNA complexes, but large excess of polycations are required for thepreparation of stable small particles.

[0211] DNA/Histone H1 Complexes.

[0212] H1 has total positive charge 55 per molecule (Mw 21.3 kDa) andcan form an inter polyelectrolyte complex with DNA. In contrast to PLLand PAA, interaction of Hi with DNA leads to considerable increase ofturbidity in broad range of H1 concentration. The turbidity is notchanged after addition of 100 mM NaCl. Treatment of H1/DNA complex withcharge ratio 3.42 by DTBP leads to significant decrease of turbidityfrom 1929 to 348. Following addition of NaCl causes the turbidity toincrease to 458.

[0213] The centrifugation of HI/DNA complexes in buffer with 100 mM NaCl7 min at 12,000 rpm results in precipitation of DNA, but after DTBPmodification most part of DNA stays in solution, which indicatespresence of small particles (FIG. 6). Table 6 shows that the sizes ofthe particles formed with DTBP (Table 6B) in 100 mM NaCl were muchsmaller that the particle formed without DTBP (Table 6A). TABLE 6 Theeffect of varying the H1DNA charge ratio on the sizes of PAA/DNAcomplexes without (A) or with (B) the addition of DTBP. The sizes weredetermined by quasi elastic light scattering and numbers indicate thepercent of particles <100 nm or >100 nm. Number in parentheses indicatethe size (diameter in nm) of the most abundant species within that sizerange. Percentage of Particles Less or Greater Than 100 nm −NaCl +NaClCharge ratio(+/−) <150 nm >150 nm <150 nm >150 nm A. H1/DNA− no DTBP1.55 7(44) 29(377)64(1376) 47(25) 36(491) 3.1 6(75) 88(500)10(6000) —92(470)8(8000) 6.2 — 10(159)90(589) — 62(350)38(1825) 9.3 9(113) 91(348)— 2(208)98(1404) B. H1/DNA+ DTBP 1.55 19(27)6(131) 75(886) —84(892)16(8000) 3.1 28(37) 12(168)60(603) — 28(171)72(512) 6.2 —75(166)25(1168) 47(55) 53(222) 9.3 48(117) 52(306) 56(75) 44(172)

[0214] Findings:

[0215] 1. DNA template polymerization of large polymers yields smallparticles that do not aggregate in physiological salt solutions.

[0216] 2. The ability to prepare small particles of condensed DNA thatdo not aggregate in a physiologic salt solution will be an extremelyuseful formulation for gene transfer and therapy.

EXAMPLE 9

[0217] Step DNA Template Polymerization Using the Comonomers of AEPD anda PEGylated-AEPD (Compound 18).

[0218] Step polymerization with DNA as a template was performed usingthe polyamine N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD) as inExample 1 except pegylated AEPD (Compound 18,N2,N2,N3,N3-tetra(PEG-amino propyl)-AEPD, or will be referred to asAEPD-PEG) was added to the reaction mixture along with AEPD. Thisexample teaches how to prepare non-aggregated, water-soluble particles(diameter <100 nm) of condensed DNA via the process of templatepolymerization.

[0219] Methods:

[0220] The mixture of the following amines was used as comonomers:

[0221] 1. N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD, Aldrich,Milwaukee, Wis.).

[0222] 2. N2,N2,N3,N3-tetra(PEG-amino propyl)-AEPD (Compound 18).

[0223] The following crosslinker was used:

[0224] 1. Dimethyl-3,3′-dithiobispropionimidate (DTBP, S-S cleavablebisimido ester, Pierce Chemical Co.).

[0225] Optimized reaction conditions with AEPD/AEPD-PEG mixture were asfollows. Plasmid DNA (pCIluc, 10 ug), AEPD (58 ug), AEPD-PEG (5 mg) andDTBP (187 ug) were mixed in 0.5 ml of buffer solution (20 mM HEPES, 1 mMEGTA, pH 8.5). Molar ratio of total AEPD (AEPD+AEPD-PEG) to DNA base was20:1. Reaction was allowed to proceed for three hours at roomtemperature. At 10, 30, 60 and 180 min time points particle sizing wasperformed using photon correlation spectrometer as described inExample 1. The data obtained was compared with the experiment where onlyAEPD was used as monomer. Three independent samples of each templatepolymerization reaction mixture were measured and the data was expressedas mean +/−SD. Transmission electron microscopy of the AEPD/AEPD-PEGmixture samples was performed as described in Example 1.

[0226] Results:

[0227] Formation of particles of condensed DNA for AEPD/AEPD-PEGtemplate polymerization mixture was confirmed both by dynamic lightscattering and by electron microscopy. Unlike AEPD alone 20:1 mixturewhich yields aggregates, AEPD/AEPD-PEG mixture resulted in increasedpopulation of non-aggregated individual DNA particles with the size <100nm of characteristic rod morphology.

[0228] 85% of the condensed DNA stays in solution after centrifugationin microcentrifuge for 5 min at 12000 g. At this conditions 100% of DNAcondensed with AEPD alone (1:20 ratio) was found precipitated.

[0229] The 1:20 DNA/AEPD/DTBP reaction mixture in the presence ofAEPD-PEG molecules with protected primary amino groups (precursor ofAEPD-PEG monomer, see Example ??) resulted in formation of aggregatedDNA.

[0230] Findings:

[0231] 1. Template polymerization can be performed in the presence ofpegylated AEPD molecules. PEG-containing monomer was included into finalcondensed DNA complex.

[0232] 2. Addition of pegylated PEG into standard DNA/AEPD/DTBP templatepolymerization mixture resulted in formation of non-aggregated particlesof condensed DNA via the mechanism of steric stabilization.

EXAMPLE 10

[0233] Chain Polymerization of Compound 11 on a DNA Template in anOrganic Solvent.

[0234] Plasmid DNA (pCI luc, 10 mg) in 660 μL water was combined with1380 μL methanol and 660 μL chloroform containing 144 mg of Compound 11,giving a clear solution with a 7 fold excess of positive charge. Thesolution was vortexed and allowed to stand at room temperature for 20minutes. One half of the monophase was reserved. The remaining monophasewas separated into two layers by the addition of an additional 375 μLwater. The two resulting layers were separated. The presence of DNA inthe chloroform layer was confirmed by absorbence at 260 nm. The sizes ofthe particles in the chloroform layer and in the reserved monophase weremeasured on a Brookhaven ZetaPlus particle sizer. The Bligh/Dyermonophase (Bligh, E. G. and Dyer, W. J. (1959) A rapid method of totallipid extraction and purification. Can. J. Biochem. Physiol., 37,911-917.) had two groups of particles in the size range of 80-128 nm and7000-10000 nm. The chloroform layer showed one group of particles with asize range of 4-7 nm, however the signal intensity was low.

[0235] The Bligh/Dyer monophase and the chloroform layer werepolymerized in the presence of 1% AIBN (Aldrich Chemical Company) for 1hour at 55° C. Particle size was measured after the polymerization. TheBligh/Dyer monophase contained one population of particles with a sizerange of 700-1000 nm. The chloroform layer contained one population ofparticles with a size range of 340-400 nm. The polymerized reactionproducts were analyzed on a SDS-PAGE gel (Novex, 10-20% tricene).Approximately 50 μg of the polymerized reactions and a controlconsisting of 7 μg DNA and 50 μg compound 11 without polymerization wereloaded onto the gel and visualized with coumassie staining. A smear ofhigh-molecular weight polymer beginning in the well was observed in bothof the polymerization reactions. The control exhibited one band of lowmolecular weight.

EXAMPLE 11

[0236] Methods:

[0237] Dextran sulfate (Mw=500 000, DS) was obtained from Sigma.Polymethacrylic acid sodium salt (PMAA, Mw=9 500) was obtained fromAldrich. Caged DNA particles were prepared as described in example“Template polymerization (caging) of large polymers” with DTBP ascross-linking agent. Zeta-potentials of the obtained particles weremeasured using Zeta-Plus Photon Correlation Spectrometer (BrookhavenInstruments Corp.). Ninety degree light scattering measurements and TOTObinding assay were performed using Fluorescence Spectrophotometer. TOTOassay was used to assess the degree of DNA condensation (Wong F M.Reimer D L. Bally M B, Cationic lipid binding to DNA: characterizationof complex formation. Biochemistry. 35(18):5756-63, 1996).

[0238] Results:

[0239] After the obtaining soluble particles of positevly-charged cagedDNA/PLL complexes their surface was rendered negatively charged bycomplexing it with the excess of polyanion. It was found that uponaddition of polyanion solution to soluble DNA/PLL complex the net chargeof the triple complex can be changed to the opposite at the certainconcentration of the polyanion TABLE 7 DS added Z-potential Z-potentialug uncaged caged  0 29.55 28.5  50 33.68 19.73 100 −17.25 −14.66 150−22.45 −13.73 200 −19.84 −18.21 300 400 500 −19.21 −14.33

[0240] Table 7. Zeta potential of caged and non-caged DNA/PLL (1:6)complexes after addition of dextran-sulfate (DS). Complexes wereprepared with 30 ug of DNA and 114 ug of PLL and caged with 240 ug ofDTBP for 2 hrs.

[0241] Triple complexes formed at 150 ug of DS were tested on solubilityat physiological salt. It was found that 60% of 190 stays in thesolution after 5 min centrifugation at 12 000 g for both caged anduncaged complexes. Particle sizing using dynamic light scatteringdemonstrated 80% particles <150 nm in diameter at these conditions.TABLE 8 TOTO signal, TOTO signal DS added, % of native DNA % of nativeDNA ug uncaged caged  0  4  3  50  3  2 100 14  7 200 49 27 300 43 22400 39 20 500 38 20

[0242] Table 8. DNA condensation after complexing DNA/PLL complexes withdextran sulfate.

[0243] TOTO assay (Table. 8) demonstrated that DNA stays condensed afterformation of negatively charged triple complex though some partialdecondensation occured. Caged complex was found more resistant todecondensation during recharging (80% condensation preserved at 400-500ug DS added).

[0244] It is possible to recharge DNA/PLL complexes with otherpolyanions. The similar data were obtained with polymethacrylic acid(not shown).

[0245] Syntheses of Compounds

[0246] Materials and Methods:

[0247] H-NMR spectra were recorded on a Bruker AC 250 or a Bruker AC 300spectrophotometer with chemical shifts given in parts per milliondownfield from an internal standard of tetramethylsilane.Diamino-N-methyldipropylamine (Aldrich Chemical Co.), Boc anhydride(Aldrich Chemical Co.), triethylamine (Aldrich Chemical Co.),trifluoroacetic anhydride (Aldrich Chemical Co.), 9-bromo-1-nonanol(Aldrich Chemical Co.), acryloyl chloride (Aldrich Chemical Co.),3-bromopropylamine hydrobromide (Aldrich Chemical Co.), 7-bromo-1-octene(Aldrich Chemical Co.), trimethylamine (25% solution in water) (AldrichChemical Co.), methyl iodide (Aldrich Chemical Co.),N,N,N′N′-tetramethyl-propane diamine (Aldrich Chemical Co.),N,N,N′,N′,N″-pentamethylethylentriamine (Aldrich Chemical Co.) were usedas supplied. All solvents were obtained from Aldrich Chemical Co. Allanhydrous solvents were obtained from Aldrich Chemical Co. inSure/Seal_containers.

[0248] 3,3′-(N′,N″-tert-butoxycarbonyl)-N-methyldipropylamine (1).3,3′-Diamino-N-methyldipropylamine (0.800 mL, 0.721 g, 5.0 mmol) wasdissolved in 5.0 mL 2.2 N sodium hydroxide (11 mmol). To the solutionwas added Boc anhydride (2.50 mL, 2.38 g, 10.9 mmol) with magneticstirring. The reaction mixture was allowed to stir at room temperatureovernight (approximately 18 hours). The reaction mixture was made basicby adding additional 2.2 N NaOH until all t-butyl carboxylic acid was insolution. The solution was then extracted into chloroform (2×20 mL). Thecombined chloroform extracts were washed 2×10 mL water and dried overmagnesium sulfate. Solvent removal yielded 1.01 g (61.7%) product as awhite solid: ¹H-NMR (CDCl₃) d 5.35 (bs, 2H), 3.17 (dt, 4H), 2.37 (t,4H), 2.15 (s, 3H), 1.65 (tt, 4H), 1.45 (s, 18H).

[0249] 3,3′-Trifluoroacetamidyl-N-methyldipropylamine (2).

[0250] 3,3′-Diamino-N-methyldipropylamine (0.504 mL, 436 mg, 3.0 mmol)and triethylamine (0.920 mL, 6.6 mmol) were dissolved in 20 mL drymethylene chloride. The solution was chilled on an ice bath.Trifluoroacetic anhydride (0.932 mL, 1.39 g, 6.6 mmol) dissolved in 40ml dry methylene chloride was added dropwise with magnetic stirring overa period of approximately 20 minutes. The reaction was allowed to cometo room temperature and to stir overnight (approximately 18 hours). Thereaction mixture was washed 2×15 mL 2% sodium bicarbonate, 2×15 mLwater, and dried over magnesium sulfate. Solvent removal yielded 763 mg(67.9%) product as a yellow oil: 1H-NMR (CDCl₃) d 8.20 (bs, 2H), 3.45(dt, 2H), 2.47 (t, 2H), 2.20 (s, 3H), 1.75 (tt, 2H).

[0251] 9-Bromononacrylate (3).

[0252] 9-Bromo-1-nonanol (0.939 g, 4.0 mmol) was dissolved in 4.0 mLanhydrous diethyl ether in a flame dried 10 mL r.b. flask under drynitrogen. Sodium carbonate (6.36 g, 6.0 mmol) was added to the reactionmixture. Acryloyl chloride (0.356 mL, 0.397 g, 4.2 mmol) dissolved in3.5 mL anhydrous ether was added dropwise over a period of approximately10 minutes. The reaction mixture was allowed to come to room temperatureand stir for two days. The reaction mixture was diluted to 40 mL withether and washed 3×10 mL 2% sodium bicarbonate to remove unreactedacryloyl chloride. The organic layer was dried over magnesium sulfateand passed through a short (approximately 7 g) alumina column to removeunreacted alcohol. Solvent removal yielded 390 mg (35.2%) product as aclear liquid: ¹H-NMR (CDCl₃) d 6.40 (dd, 1H), 6.12 (dd, 1H), 5.82 (dd,1H), 4.15 (t, 4H), 3.40 (t, 2H), 1.85 (dt, 2H), 1.65 (dt, 2H), 1.35 (m,10H).

[0253] 3-Bromo-1-(trifluoroacetamidyl)propane (4).

[0254] 3-Bromopropylamine Hydrobromide (2.19 g, 10.0 mmol) andtriethylamine (1.67 mL, 12.0 mmol) were dissolved in 60 mL dry methylenechloride. The solution was chilled on an ice bath. Trifluoroaceticanhydride (1.69 mL, 2.51 g, 12.0 mmol) dissolved in 60 mL dry methylenechloride was added dropwise over approximately 20 minutes. The reactionwas allowed to come to room temperature and was stirred overnight(approximately 18 hours). The reaction mixture was washed 1×10 mL 2%sodium bicarbonate, 1×10 mL water, and dried over magnesium sulfate.Solvent removal yielded 2.07 g (88.5%) product as a white powder: ¹H-NMR(CDCl₃) d 6.70 (bs, 1H), 3.55 (dt, 2H), 3.45 (t, 2H), 2.17 (tt, 2H).

[0255] 1-Octene-7-trimethylammonium Bromide (5).

[0256] 7-Bromo-1-octene (0.168 mL, 191.2 mg, 1.00 mmol) was combinedwith trimethylamine (2.40 mL 25% solution in water). The mixture wasincubated at 50 C_ on a rotary shaker for 18 hours. Solvent removal andrecrystalization from acetone/diethyl ether yielded 191.6 mg (76.6%)product as white plates: ¹H-NMR (CDCl₃) d 5.75 (m, 1H), 5.00 (m, 2H),3.60 (m, 2H), 3.45 (s, 9H), 2.05 (m, 2H), 1.75 (m, 2H), 1.40 (m, 6H).

[0257]3,3′-(N′,N″-tert-butoxycarbonyl)-N-(7-octene)-N-methyldipropyl-ammoniumBromide (6). Compound 1 (86.3 mg, 0.25 mmol) was combined with7-bromo-1-octene and dissolved in 0.050 mL methyl sulfoxide. Thereaction mixture was incubated at 55 C_for 18 hours. The viscousreaction mixture was triturated with ether twice. The remaining oil wasrecrystalized from chloroform/ether to yield 55.3 mg (48.7%) product aswhite crystals: ¹H-NMR (CDCl₃) d 5.75 (m, 3H), 4.95 (m, 2H), 3.55 (m,4H), 3.30 (m, 6H), 3.15 (s, 3H), 2.05 (m, 4H), 1.97 (m, 2H), 1.70 (m,2H), 1.45 (s, 18H), 1.35 (m, 6H).

[0258] 3,3′-diamine-N-(7-octene)-N-methyldipropylammonium Bromide (7).

[0259] Compound 6 was combined with 0.350 mL ethyl acetate, 0.150 mLmethanol, and 0.150 mL 12 N hydrochloric acid. The reaction mixture wasstirred at room temperature for 2.5 hours, during this time the reactionbecame homogenous. Solvent was removed and the product was precipitatedfrom a small amount of methanol with ether to yield 36.0 mg (95.2%)product as a colorless oil: ¹H-NMR (CD₃OD) d 5.85 (m, 1H), 5.00 (m, 2H),3.55 (m, 4H), 3.45 (m, 2H), 3.20 (s, 3H), 3.15 (t, 4H), 2.25 (m, 4H),2.10 (m, 2H) 1.85 (m, 2H), 1.50 (m, 6H).

[0260] 3,3′-(N′,N″-tert-butoxycarbonyl)-N,N-dimethyldipropylammoniumbromide (8). Compound 1(75.0 mg, 0.217 mmol) was dissolved in 0.5 mL dryether, ethyl alcohol was added drop-wise until compound 1 was completelydissolved. The reaction solution was chilled on an ice bath and purgedwith dry nitrogen. Methyl iodide (0.021 mL, 33.7 mmol) was added, andthe reaction mixture was stirred at 4 C_for 18 hours. Poured reactionmixture into water and washed with ether. After removal of water theproduct was dissolved in chloroform, decolorized with activatedcharcoal, and dried with magnesium sulfate. Solvent removal yielded 92.0mg (87.0%) product as a yellow oil: ¹H-NMR (CDCl₃) d 5.50 (bs, 2H), 3.60(m, 4H), 3.30 (s, 6H), 3.25 (m, 4H), 2.07 (m, 4H), 1.45 (s, 18H).

[0261] 3,3′-Diamino-N,N-dimethyldipropylammonium Bromide (9).

[0262] Compound 8 (92.0 mg, 0.189 mmol) was dissolved in 0.200 mL ethylacetate and 0.150 mL 12 N hydrochloric acid. The reaction mixture wasstirred at room temperature for 1 hour. Solvent was removed and the oilyresidue was triturated three times with ether. The remaining product wasdried in vacuo yielding 43.9 mg (100%) product as a yellow waxy solid:H-NMR (CD₃OD) d 3.55 (m, 4H), 3.20 (s, 6H), 3.20 (t, 4H), 2.22 (m, 4H).

[0263] N,N′-Dinonacrylate-N,N,N′,N′-tetramethylpropanediammonium bromide(10).

[0264] N,N,N′N′-tetramethylpropane diamine (0.0252 mL, 0.15 mmol) andcompound 3 (131 mg, 0.148 mmol) were dissolved in 0.150 mLdimethylformamide. The reaction mixture was incubated at 50 C_ for 5days. The product was precipitated from the reaction mixture by theaddition of ether. The resulting solid was collected and recrystalizedtwice from ethanol/ether yielding 56.9 mg (55.4%) product as whitecrystals: ¹H-NMR (CDCl₃) d 6.40 (dd, 2H), 6.15 (dd, 2H), 5.85 (dd, 1H),4.15 (t, 4H), 3.88 (m, 4H), 3.52 (m, 4H), 3.40 (s, 12H), 2.75 (m, 2H),1.82 (m, 4H), 1.65 (m, 4H), 1.35 (m, 20H).

[0265]N,N′,N″-Trinonacrylate-N,N,N′,N′,N″-pentamethyldiethylentriammoniumBromide (11). N,N,N′,N′,N″-pentamethylethylentriamine (0.031 mL, 0.15mmol) and compound 3 (187 mg, 0.675 mmol) were dissolved in 0.150 mLdimethylformamide. The reaction mixture was incubated at 50 C_ for 5days. The product was precipitated from the reaction mixture by theaddition of ether. The resulting solid was collected and recrystalizedfrom ethanol/ether yielding 36.6 mg (24.3%) product as white crystals:H-NMR (CDCl₃) d 6.40 (dd, 3H), 6.15 (dd, 3H), 5.83 (dd, 3H), 4.15 (t,6H), 3.95 (m, 4H), 3.60 (m, 4H), 3.40 (s, 15H), 3.17 (m, 6H), 1.70 (m,12H), 1.35 (m, 30H).

[0266] 3,3′-Trifluoroacetamidyl-N-nonacrylate-N-methyldipropylammoniumBromide (12).

[0267] Compound 2 (233 mg, 0.691 mmol and compound 3 (282 mg, 1.01 mmol)were dissolved in 0.200 mL dimethylformamide. The reaction mixture wasincubated at 50 C_for 4 days. The product was precipitated from thereaction mixture by the addition of ether. The resulting oil wastriturated 3× with ether. The oil was dried in vacuo yielding 385.5 mg(90.8%) product as a clear oil: ¹H-NMR (CDCl₃) d 9.05 (bs, 2H), 6.35(dd, 1H), 4.15 (t, 2H), 3.50 (m, 8H), 3.20 (m, 2H), 3.15 (s, 3H), 2.20(m, 4H), 1.70 (m, 4H), 1.30 (m, 10H).

[0268]3,3′-(N′,N″-tert-butoxycarbonyl)-N-(3′-trifluoroacetamidylpropane)-N-methyldipropylammoniumbromide (13).

[0269] Compound 1 (100.6 mg, 0.291 mmol) and compound 4 (76.8 mg, 0.328mmol) were dissolved in 0.150 mL dimethylformamide. The reaction mixturewas incubated at 50 C_ for 3 days. TLC (reverse phase; acetonitrile: 50mM ammonium acetate pH 4.0; 3:1) showed 1 major and 2 minor spots noneof which corresponded to starting material. Recrystalization attemptswere unsuccessful so product was precipitated from ethanol with etheryielding 165.5 mg (98.2 %) product and minor impurities as a clear oil:¹H-NMR (CDCl₃) d 9.12 (bs, 1H), 5.65 (bs, 2H), 3.50 (m, 8H), 3.20 (m,4H), 3.15 (s, 3H), 2.20 (m, 2H), 2.00 (m, 4H), 1.45 (s, 18H).

[0270]3,3′-(N′,N″-tert-butoxycarbonyl)-N-(3″-aminopropane)-N-methyl-dipropylammoniumbromide (14). Compound 13 (1.09 g, 1.88 mmol) was dissolved in mLmethanol and 1.0 mL water. Sodium carbonate (1.00 g, 9.47 mmol) wasadded, and the reaction mixture was stirred at room temperature for 18hours. Sodium carbonate and solvent were removed leaving a clear oilwhich was triturated 3× with ether. acuum drying yielded 898.2 mg(98.8%) product as a white solid. TLC (reverse phase; acetonitrile: 50mM ammonium acetate pH 4.0; 1:3) gave 1 spot rf =0.54. ¹H-NMR (D20) d3.55 (m, 6H), 3.27 (m, 4H), 3.05 (s, 3H), 2.87 (m, 2H), 1.97 (m, 6H),1.45 (s, 18H).

[0271] N₁,N₄-(tert-butoxycarbonyl)-bis(2-aminoethyl)-1,3-propanediamine(15)

[0272] AEPD (275 mg, 1.72 mmol) was dissolved in 5.0 mL tetrahydrofuranand chilled to 0° C. on an ice bath. BOC-ON (800 mg, 3.25 mmol, AldrichChemical Co.) dissolved in 3 mL tetrahydrofuran was added dropwise withmagnetic stirring over approximately 15 minutes. The ice bath wasremoved and the stirring reaction mixture was allowed to come to roomtemperature. After 2 hours the solvent was removed on a rotaryevaporator, and the residue was dissolved in 20 mL chloroform. Thechloroform was washed with 2 N sodium hydroxide. The chloroform layerwas then extracted with 0.1 N hydrochloric acid. The acid layer was thenmade basic by the addition of 2 N sodium hydroxide and the product wasback extracted into chloroform. The chloroform was dried over magnesiumsulfate. Solvent removal afforded 288 mg product (49.2%). ¹H-NMR (CD₃OD)∂ 3.15 (t, 4H), 2.65 (m, 8H), 1.70 (m, 2H), 1.45 (s, 18H).

[0273]N₂,N₂,N₃,N₃-(3′-trifluoroacetamidylpropane)-N₁,N₄-(tert-butoxycarbonyl)-bis(2-aminoethyl)-1,3-propanediammoniumDibromide (16)

[0274] Compound 15 (33.0 mg, 91.7 μmol) and3-Bromo-1-(trifluoroacetamidyl)propane (128 mg, 547 μmol) were combinedin 200 μL dimethylformamide and incubated at 55° C. for 24 hours. TLC(silica: 90% acetonitrile, 10% 50 mM ammonium acetate pH 4.0) showed amixture of products. Additional 3-Bromo-1-(trifluoroacetamidyl)propane(100 mg, 427 μmol) in 200 μL dimethylformamide and incubated anadditional 24 hours. TLC showed 2 spots at rf of 0.51 and 0.58 in theabove system when developed with dragendorffs reagent (Sigma ChemicalCo.) Precipitation with diethyl ether yielded 56.0 mg product (53%). Thefinal product may be a mixture of tri-alkylated AEPD and tetra-alkylatedAEPD. Full characterization and purification is in progress.

[0275]N₂,N₂,N₃,N₃-(3′-aminopropane)-N₁,N₄-(tert-butoxycarbonyl)-bis(2-aminoethyl)-1,3-propanediammoniumdi-trifluoroacetate (17)

[0276] Compound 16 (28.0 mg, 24.7 μmol) dissolved in 1 mL 6:4 methanolwater along with calcium carbonate (104 mg, 1.0 mmol). The reactionmixture was stirred at 60° C. for 3 hours. TLC (silica: 90%acetonitrile, 10% 50 mM ammonium acetate pH 4.0) indicated completion ofreaction with all material remaining at the origin. Product was isolatedafter removal of calcium carbonate by filtration to yield 13.0 mg(75.1%).

[0277]N₂,N₂,N₃,N₃-(3′-PEG₅₀₀₀aminopropane)-N₁,N₄-bis(2-aminoethyl)-1,3-propanediammoniumDi-trifluoroacetate (18)

[0278] Compound 17 (13 mg, 18.6 μmol) and0-[2-(N-succinimidyloxycarbonyl)-ethyl]-O′-methylpolyethylene glycol5,000 [NHS-Peg] (180 mg, 36 μmol) in 0.5 ML dimethylformamide. Thereaction was stirred for 30 minutes. The reaction mixture was checkedfor the presence of primary amines by spotting on TLC plate anddeveloping with ninhydrin spray. Primary amines were still present soadditional NHS-Peg (80 mg, 16 μmol) was added. The reaction mixture wasagain screened for the presence of primary amines, none were found to bepresent. The reaction was stopped by precipitation with diethyl ether.The precipitate was washed 2× with diethyl ether, and dried under vacuumto yield 198 mg product. The product was dissolved in 2 mLtrifluoroacetic acid, and incubated 20 minutes to remove the BOCprotecting groups. The trifluoroacetic acid was removed under a streamof nitrogen. The residue was dried under vacuum to yield 198 mg productas an off-white solid. The presence of free amino groups after theremoval of the BOC protecting groups was confirmed by a positiveninhydrin test. The final product should contain approximately 3 Pegchains per molecule as determined by the amount of NHS-Peg used inreaction.

[0279] The foregoing is considered as illustrative only of theprinciples of the invention. Furthermore, since numerous modificationsand changes will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationshown and described. Accordingly, all suitable modifications andequivalents fall within the scope of the invention.

We claim:
 1. A method of making a compound for delivery to a cell,comprising: forming a polymer in the presence of a biologically activedrug.
 2. The method of claim 1 wherein the drug consists of a polyion.3. The method of claim 2 further comprising modifying the polymer in thepresence of the polyion.
 4. The method of claim 3 further comprisingmodifying the polymer by attaching a molecule.
 5. The method of claim 4wherein the molecule consists of a gene transfer enhancing signal. 6.The method of claim 5 wherein the gene transfer enhancing signal isselected from the group consisting of a nuclear localizing signal, aligand that binds a cell, and a releasing signal.
 7. The method of claim4 wherein the molecule changes the charge of the polymer.
 8. The methodof claim 4 wherein the molecule is selected from the group consisting ofamphipathic, hydrophobic and hydrophilic compounds.
 9. The method ofclaim 3 wherein the polymer is modified by mixing the polymer with amolecule.
 10. The method of claim 9 wherein the molecule consists of agene transfer enhancing signal.
 11. The method of claim 9 wherein themolecule changes the charge of the compound.
 12. The method of claim 9wherein the molecule is selected from the group consisting ofamphipathic, hydrophobic and hydrophilic compounds.
 13. The method ofclaim 3 wherein the polymer contains a disulfide bond.
 14. A method ofmaking a compound for delivery to a cell, comprising: a) cross-linking apolymer in the presence of a polyion, thereby forming a complex ofpolymer and polyion; and, b) delivering the complex to the cell.
 15. Themethod of claim 14 wherein the polymer is selected from the groupconsisting of polycations and polyanions.
 16. The method of claim 15further comprising attaching a molecule to the polymer.
 17. The methodof claim 16 wherein the molecule consists of a gene transfer enhancingsignal.
 18. The method of claim 17 wherein the gene transfer enhancingsignal is selected from the group consisting of a nuclear localizingsignal, a ligand that binds a cellular receptor, and a releasing signal.19. The method of claim 16 wherein the molecule is selected from thegroup consisting of amphipathic, hydrophobic and hydrophilic compounds.20. The method of claim 14 further comprising: mixing the complex with amolecule.
 21. The method of claim 20 wherein the net charge of thecomplex is changed.
 22. A method of making a compound for delivery to acell, comprising: modifying a molecule in the presence of the polyionthereby providing a deliverable polyion.
 23. A monomer for forming apolymer having the general structure comprising:

R is selected from the group consisting of a steric stabilizer andhydrogen; R′ is selected from the group consisting of a stericstabilizer and hydrogen; R″ is selected from the group consisting of asteric stabilizer and hydrogen; R′″ is selected from the groupconsisting of a steric stabilizer and hydrogen; a is selected from thegroup consisting of 1-20; b is selected from the group consisting of1-20; c is selected from the group consisting of 1-20; and, X⁻ consistsof a monovalent anion.
 24. A monomer for forming a polymer having thegeneral structure comprising:

wherein, R consists of protecting group 1; R′ consists of orthogonalprotecting group 2; and, X⁻ consists of monovalent anion.
 25. A monomerfor forming a polymer having the general structure comprising:

R is selected from the group consisting of orthogonal protectingmolecules 1, targeting molecules, steric stabilizers, and hydrogen; R′is selected from the group consisting of orthogonal protecting molecules2, targeting molecules, steric stabilization molecules, and hydrogen. ais selected from the group consisting of 1-20; b is selected from thegroup consisting of 1-20; c is selected from the group consisting of1-20; d is selected from the group consisting of 1-20; and X− consistsof a monovalent anion.
 26. The method of claim 14 wherein the complex isin a solution of changeable tonicity.
 27. A method of making a compoundfor delivery to a cell, comprising: mixing a polyion with a firstpolymer and a second polymer thereby forming a deliverable complex.